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>HYSIOLOGICAL STUDIES OF BACILLUS 
RADICICOLA OF CANADA 
FIELD PEA 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 



MARTIN J.°PRUCHA 



JUNE, 1913 



(Reprinted from Memoir No. 5, March, 1915, of Cornell University Agricultural Experi- 
ment Station as Contribution No. 15 of the Laboratory of Plant Physiology) 



PHYSIOLOGICAL STUDIES OF BACILLUS 

RADICICOLA OF CANADA 

FIELD PEA 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 
5^ 



MARTIN J. PRUCHA 



JUNE, 1913 



(Reprinted from Memoir No. 5, March, 1915, of Cornell University Agricultural Experi- 
ment Station as Contribution No. 15 of the Laboratory of Plant Physiology) 



\D 






In exchange 
Cornell Univ. Library 

DEC 8 - 1915 



CORNELL UNIVERSITY 
AGRICULTURAL EXPERIMENT STATION 

Experimenting Staff 

BEVERLY T. GALLOWAY, B.Agr.Sc, LL.D., Director. 

ALBERT R. MANN, B.SA., Secretary. 

JOHN H. COMSTOCK, B.S., Entomology. 

HENRY H. WING, M.S. in Agr., Animal Husbandry. 

T. LYTTLETON LYON, Ph.D., Soil Technology. 

JOHN L. STONE, BAgr., Farm Practice. 

JAMES E. RICE, B.S.A., Poultry Husbandry. 

GEORGE W. CAVANAUGH, B.S., Agricultural Chemistry. 

HERBERT H. WHETZEL, M.A., Plant Pathology. 

ELMER O. FIPPIN, B.S.A., Soil Technology. 

G. F. WARREN, Ph.D., Farm Management. 

WILLIAM A. STOCKING, Jr., M. S.A., Dairy Industry. 

CHARLES S. WILSON, A.B., M.S.A., Pomology 

WILFORD M. WILSON, M.D., Meteorology. 

RALPH S. HOSMER, M.F., Forestry. 

JAMES G. NEEDHAM, Ph.D.; Entomology and Limnology. 

ROLLINS A. EMERSON, Ph.D., Plant Breeding. 

HARRY H. LOVE, Ph.D., Plant Breeding. 

ARTHUR W. GILBERT, Ph.D., Plant Breeding. 

DONALD REDDICK, Ph.D., Plant Pathology. 

EDWARD G. MONTGOMERY, M.A., Farm Crops. 

WILLIAM A. RILEY, Ph.D., Entomology. 

MERRITT W. HARPER, M.S., Animal Husbandry. 

J. A. BIZZELL, Ph.D., Soil Technology. 

GLENN W. HERRICK, B.S.A., Economic Entomology. 

HOWARD W. RILEY, M.E., Farm Mechanics. 

CYRUS R. CROSBY, A.B., Entomology. 

HAROLD E. ROSS, M.S.A., Dairy Industry. 

KARL McK. WIEGAND, Ph.D., Botany. 

EDWARD A. WHITE, B.S., Floriculture. 

WILLIAM H. CHANDLER, M.S. in Agr., Pomology. 

ELMER S. SAVAGE, M.S.A., Ph.D., Animal Husbandry. 

LEWIS KNUDSON, Ph.D., Plant Physiology. 

KENNETH C. LIVERMORE, B.S. in Agr., Farm Management. 

ALVIN C. BEAL, Ph.D., Floriculture. 

MORTIER F. BARRUS, Ph.D., Plant Pathology. 

CLYDE H. MYERS, M.S., Ph.D., Plant Breeding. 

GEORGE W. TAILBY, Jr., B.S.A., Superintendent of Live Stock. 

EDWARD S. GUTHRIE, M.S. in Agr., Ph.D., Dairy Industry. 

JAMES C. BRADLEY, Ph.D., Entomology. 

PAUL WORK, B.S., A.B., Vegetable Gardening. 

JOHN BENTLEY, Jr., B.S., M.F., Forestry. 

EARL W. BENJAMIN, Ph.D., Poultry Husbandry. 

EMMONS W. LELAND, B.S.A., Soil Technology. 

CHARLES T. GREGORY, Ph.D., Plant Pathology. 

WALTER W. FISK, M.S.A., Dairy Industry. 

ARTHUR L. THOMPSON, M.S. in Agr., Farm Management 

ROBERT MATHESON, Ph.D., Entomology. 

HORACE M. PICKERILL, B.S., Dairy Industry. 

MORTIMER D. LEONARD, B.S., Entomology. 

FRANK E. RICE, Ph.D., Agricultural Chemistry. 

V. B. STEWART, Ph.D., Plant Pathology. 

BRISTOW ADAMS, B.A., Editor. 

LELA G. GROSS, Assistant Editor. 

The regular bulletins of the Station are sent free on request to residents of New York State. 

3 



CONTENTS 

PAGE 

Introduction 9 

Scope of the investigation 9 

Method of investigation 10 

Organism 10 

Media 10 

Sterilization of utensils 10 

Sterilization of seed 10 

Sterilization of media and soil 12 

Method of growing the plants 12 

Inoculations 13 

Examination of plants for nodules 13 

Special method for growing plants under sterile conditions. . 13 

Part I. Isolation and identification of the organism 14 

Isolation of Bacillus radicicola 14 

Identification of the organism 15 

Morphology of the organism 16 

Cultural and biochemical features 18 

Part II. Influence of certain factors on nodule production 19 

Experiment 1. Influence of aeration in light and in darkness. . . . 21 
Experiment 2. Influence of some nutrient solutions in light and 

in darkness 22 

Experiment 3. Influence of potassium nitrate in light and in 

darkness 24 

Experiment 4. Influence of potassium nitrate and calcium nitrate 

in Pfeffer's solution 25 

Experiment 5. Influence of various concentrations of Pfeffer's 

solution, both with and without nitrates 26 

Experiment 6. Influence of Pfeffer's solution in which the essen- 
tial elements were absent 26 

Experiment 7. Influence of moisture 27 

Experiment 8. Influence of certain substances in varying 

quantities 27 

Experiment 9. Influence of certain additional substances 29 

General discussion of results of experiments 32 

5 



6 Contents 

PAGE 

Part III. Influence of various media on the infecting power and the 

vitality of Bacillus radicicola 32 

Experiment 10. Influence of clay, loam, sand, and carborundum 39 
Experiment 11. Influence of hydrochloric acid, sodium hy- 
droxide, and cane sugar, in varying concentrations 41 

Experiment 12. Influence of some other media 47 

Experiment 13. Influence of media 300, 310, 335, and 400 68 

Summary 73 

General discussion 75 

Acknowledgments 78 

Bibliography 79 



PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA 
OF CANADA FIELD PEA 



PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA 

OF CANADA FIELD PEA 1 

Martin J. Prucha 

INTRODUCTION 

Ever since the discovery that the formation of nodules on the roots of 
legumes is associated with a definite microorganism, there have appeared 
on the market pure cultures of this organism for the artificial inoculation 
of leguminous plants. Nobbe and Hiltner (1897) 2 first caused to be 
placed on the market a pure culture called nitragin, 3 in which the medium 
employed was gelatin. The efficiency of the culture was low, and Nobbe 
and Hiltner explained this by ascribing to the culture a loss of infecting 
power due to the medium used. Moore (1905) stated, as a result of his 
work, that the virulence of the organism was impaired when it was prop- 
agated on nitrogenous media, and consequently he used a nitrogen-free 
medium. 

The same opinion has been held by various other investigators, and 
is observed by all firms and institutions that distribute pure cultures 
of the legume organism. Despite the fact that media low in nitrogen 
have been employed, the results with pure cultures have not always 
been satisfactory, and Kellerman (1912) has stated that the pure 
cultures have not been as efficient as field soil. It would seem, then, that 
the conditions of isolation and cultivation of the legume organism must 
influence the organism, or that possibly the soil conditions would exert 
some influence on the efficiency of the organism to effect inoculation or 
would have an influence on the plant to resist infection. 

SCOPE OF THE INVESTIGATION 

With these ideas in mind, the investigation herewith reported was 
undertaken. It is concerned with the following: (l) Isolation and iden- 
tification of the organism causing nodule development on the roots of 
Canada field pea; (2) A study of the influence of various factors on nodule 

1 Laboratory of Plant Physiology, Contribution No. 15. 

2 Dates in parenthesis refer to bibliography, page 79. 

3 This is not the nitragin now on the market.^ 



10 Martin J. Prucha 

development in Canada field pea when the plant is grown in water or 
soil cultures; (3) A study of the influence of various environmental con- 
ditions on the infecting power of the organism. 

METHOD OF INVESTIGATION 

Organism 
Bacillus radicicola from Canada field pea was used for all the experiments 
except number 11, for which the organism from alfalfa was used. The 
organism was isolated as described on page 14. The stock culture was 
kept in the laboratory on agar slopes of medium 335, and was exposed 
to diffused light during the entire investigation. It was transferred at 
irregular intervals of time varying from one to three months. 

Media 
The various kinds of media used are referred to in the text by their 
laboratory numbers. In table 1 are given the laboratory numbers and 
the composition of the principal media employed. In addition to these 
a number of other media were used, and these are described subsequently. 
All the media were prepared according to the recommendations on the 
Descriptive Chart of the Society of American Bacteriologists. 

Sterilization of utensils 

Strict precautions were observed in sterilizing all the utensils. Glassware 
was sterilized for two hours at from 150° to 170° C. Scalpels and similar 
instruments, which resist high temperature, were heated in the flame. 
For work requiring air free from dust, a room was used into which a 
stream of steam was allowed to flow until the air was saturated; after 
condensation of the steam, the air in the room was conspicuously free 
from any dust. 

Sterilization of seed 

The experiments in Part III required seed free from B. radicicola. It 
was necessary, therefore, to sterilize the seed. In experiments 10 and 11, 
95-per-cent alcohol was used, the seed being treated for thirty seconds. 
In experiments 12 and 13 a solution of bleaching powder was used. The 
author is indebted for this method to Professor J. K. Wilson, of Cornell 
University. The solution is prepared by mixing 8 grams of bleaching 



Studies of Bacillus Radicicola of Canada Field Pea 11 
TABLE 1. Laboratory Numbers, Composition, and Reaction of Media 



Laboratory number 


Composition 


Reaction 


Medium 101 


0.2 gram MgSO, 


. 




1 gram KH-PO4 






0.3 gram Ca (NO,)' 


■ Not changed 




0.01 gram Fed, 




1000 cc. distilled water 








' 


Medium 300 


15 grams agar 


\ 




3 grams Liebig's beef extract 






10 grams Witte's peptone 

1000 cc. distilled water 


\ +1, Fuller's scale 








Medium 310 


Same as 300, plus 2 per cent dextrose 


Neutral 


Medium 331 


15 grams agar 


) 




10 grams cane sugar 






1 gramKPLPCh 


!■ Not changed 




0.2 gram MgSO^ 










Medium 334 


15 grams agar 


1 




0.2 gram K 2 HPO ; 






0.2 gram MgSO 






0.2 gram NaCl 


• Not changed 




0.2 gram CaSO s 




0.2 gram CaCO:, 






1000 cc. tap water 








' 


Medium 335 


Same as 334, plus 20 grams cane sugar 


Not changed 


Medium 337 


Same as 335, plus 10 grams Witte's peptone.. . 


Not changed 


Medium 400 


15 grams agar 


1 




3 grams Liebig's beef extract 






20 grams Witte's peptone 


[ +1, Fuller's scale 




1000 cc. distilled water 










Crone's solution .... 


1 gram KNO3 


1 




0.5 gram Fe 3 (PCh), 




. 25 gram CaSOj (gypsum) 


\ Not changed 




2000 cc. distilled water 


1 








Pfeffer's solution. . . . 


4 grams CatNO,). 


^ 




1 gram KN0 3 






1 gram MgS0 4 






. 5 gram KC1 


!• Not changed 




0.06 gram FeCh 






6000 cc. distilled water 











12 Martin J. Prucha 

powder with 140 cubic centimeters of water; after standing for a few 
hours, the clear liquid is decanted and is then ready for use. Seed was 
kept for three hours in this solution. According to Professor Wilson's 
results, seed treated in this way is completely sterilized. This method 
of seed sterilization will later be published in detail. 

That the methods of seed sterilization employed were effective was 
proved by a large number of experiments, and particularly by experiments 
with soy beans. The organism producing nodules in soy bean is not 
present in the soil of this region, and in all the experiments made not a 
single nodule developed in the cultures in which sterilized seed was used 
unless the plants were subsequently inoculated. Unsterilized seed oc- 
casionally produced plants with nodules. 

Sterilization of media and soil 

All the media used for pure cultures, if in test tubes of small volume, 
were sterilized in an autoclave for fifteen minutes at 120° C. 

In experiments 10, 11, 12, and 13 it was necessary to sterilize the soil 
in which the plants were grown. Three-inch flowerpots and glass tum- 
blers, each containing about 300 grams of soil, were used for this purpose. 
These were sterilized for three hours at 120° C, in a large canner's retort. 
This retort was found very useful, since several hundred of the flowerpots 
could be sterilized at one time. 

Method of growing the plants 

In experiments 1, 2, 3, 4, 5, and 6, the plants were grown in water 
cultures; in experiments 7, 8, 9, 10, 11, 12, and 13 they were grown in soil. 

For the water cultures glass vessels were employed. The vessels were 
filled with the nutrient solution and the opening was covered with par- 
affin paper. The seed was then germinated in a moist chamber, and 
when the radicle was about thiee centimeters long it was inserted into 
the solution through a small hole in the paraffin paper, allowing the coty- 
ledons to rest on top of the paper. 

For the soil cultures flowerpots and glass tumblers were used. These 
were filled with sandy soil, were covered with paper, and, for experiments 
10, 11, 12, and 13, were sterilized. The seed was planted directly in the 
soil, the paper covers being kept on until the seedlings began to push 
them off. 



Studies of Bacillus Radicicola of Canada Field Pea 13 

Distilled water was used for watering the plants in experiments 1, 2, 3, 
4, 5, 6, 7, 8, and 9. In experiments 10, 11, 12, and 13, boiled tap water 
was used. 

The plants in all the experiments were grown in the greenhouse. When 
special precautions were necessary to guard against contamination during 
the growing period, the plants were kept in an especially constructed 
culture room. For this purpose a part of the greenhouse was set off by 
a partition. Cracks in the walls and around the panes of glass were 
filled with plaster of paris. For ventilation, two panes of glass were 
replaced by a special frame fitted with a layer of cotton held between 
two pieces of cheesecloth. 

Inoculations 

Inoculations were made at the time of planting the seeds or within a 
day or two following. The culture of the organism to be used for in- 
oculation was introduced into sterile water, and the infusion was then 
added to the soil or the water culture in which the plants were grown. 
In some experiments quantitative inoculation was made, in which a spe- 
cific amount of the infusion was added. 

Examination of plants for nodules 
Nodules usually appeared in about two weeks. The plants were exam- 
ined at the end of three weeks. The roots were washed and the nodules 
were counted, and a note was made of the size and the place of attachment 
of the nodules. Since the soil in the vicinity of Ithaca is well inoculated 
with the Canada field pea organism, plants kept longer than three weeks 
were subject to some contamination. 

Special method for growing plants under sterile conditions 
For certain experiments it was necessary to maintain absolutely sterile 
conditions throughout the period of experiment. The method employed 
was as follows: A large glass cylinder, 65 centimeters high and 
10 centimeters in diameter, was used as a growth chamber. In the bottom 
of the cylinder a few pieces of broken flowerpot were placed and were 
just covered with water, and on the top of these was set a four-inch pot, 
filled with a sandy soil. The cylinder was plugged with cotton, through 
which was passed a glass tube 7 millimeters in diameter and 65 centi- 
meters long, the lower end resting on the surface of the soil in the pot 



14 Martin J. Prucha 

and the upper end protruding above the cotton plug. The tube was 
plugged at its upper end with cotton. The whole was sterilized in the 
autoclave for five hours at 15 pounds pressure. 

The seeds were sterilized by the bleaching-powder method. The steri- 
lized seed were dropped into the pots through the glass tube, and by 
manipulation of the tube they were buried in the soil. The soil of the 
pots was inoculated by introducing through the tube a few cubic centi- 
meters of water containing the nodule-forming organisms. 

PART I. ISOLATION AND IDENTIFICATION OF THE ORGANISM 
ISOLATION OF BACILLUS RADICICOLA 

On October 10, 1910, a field pea plant 30 centimeters high, with a great 
abundance of nodules, was procured. The whole plant was washed 
thoroughly in running water. One of the nodules, of firm consistency, 
was selected and cut off in such a way as to leave about 3 centimeters of 
the root on each side of the nodule; cut off in this way the nodule is more 
easily manipulated. The nodule was then disinfected in a solution of 
one part of formalin to forty parts of water, for five minutes. Four 
petri dishes were prepared, each containing a few drops of sterile water. 
The nodule, after being disinfected, was washed in sterile water, placed 
on a filter paper, and cut open, and with a pointed scalpel a part of the 
central tissue was removed and placed in petri dish 1. This nodule 
tissue was crushed and mixed with the water. Three loopfuls of this 
infusion was transferred from plate 1 to plate 2, three loopfuls from 
plate 2 to plate 3, and three loopfuls from plate 3 to plate 4. Ten cubic 
centimeters of medium 331 were then poured into each petri dish, and after 
sufficient agitation to effect equal distribution of the organisms the plates 
were allowed to incubate at 20° C. In three days a few colonies charac- 
teristic of the nodule organism became visible on plate 1; on the other 
plates plenty of colonies were present, but were visible only by the micro- 
scope. In ten days the small colonies became large enough to be con- 
veniently transferred. 

As far as could be ascertained from the general appearance of the colo- 
nies, all the plates contained only one organism. The large colonies that 
developed on plate 1 appeared to be giant colonies, having started from 
small pieces of the nodule tissue in which a large number of the organisms 
were held. In order to obtain a pure culture of this organism, one of the 



Studies of Bacillus Radicicola of Canada Field Pea 15 

small colonies was introduced into 5 cubic centimeters of sterile water in 
a test tube, and from this infusion a number of petri dishes of different 
dilutions were again prepared. From one of the colonies that developed 
on these petri dishes a transfer was made on agar slope with medium 335. 
This culture was used in all the experiments except experiment 11. The 
stock culture was kept on a shelf in the laboratory, and consequently 
was exposed for nearly three years to diffused light and to the ordinary 
variations of temperature and other atmospheric changes. The trans- 
ferring of the stock culture was made at irregular intervals of time varying 
from one to three months, and the cultures were kept in test tubes on 
agar slopes of medium 335. 

identification of the organism 

In order to be certain that the organism isolated was the causal organism 
of the nodules on Canada field peas, the following procedure was followed : 
(1) Canada field peas were grown under sterile conditions and were inoc- 
ulated with this organism; (2) from one of the nodules that developed 
under sterile conditions a culture, No. 2, was isolated by the same method 
as described above; (3) the original culture and the culture No. 2 were 
again tested as to their ability to cause the development of nodules on 
Canada field peas under sterile conditions; (4) the two cultures were 
compared in the laboratory with respect to their morphological characters 
and physiological activities. 

Ten Canada field peas were grown under sterile conditions according to 
the method described on page 13, one plant in each of ten glass cylinders. 
Five of the plants were inoculated with the above organism, and five 
were left without inoculation, as controls. At the end of six- weeks the 
plants were examined. They had made a fair growth, having reached a 
height of 60 centimeters. They were spindling, however, the leaves were 
small, and the root system was very poorly developed. All the inoculated 
plants had nodules on their roots, while the controls were free from any 
nodules. Each plant and the soil in which it was grown were examined 
for contamination. One of the controls was found to be contaminated 
with a mold, and one of the inoculated plants was contaminated with a 
yellow organism. Four of the controls were sterile, and four of the in- 
oculated plants were found to be sterile with respect to organisms other 
than B. radicicola. 



16 Martin J. Prucha 

A pure culture of the organism was isolated from a nodule found on one 
of the plants grown under sterile conditions. This organism was again 
tested as to its ability to produce nodules on Canada field peas. Fifteen 
Canada field pea plants were grown under sterile conditions again, in a 
similar manner to that described above. Five were inoculated with this 
organism, five were inoculated with the original organism, and five were 
left as controls. Again all the plants inoculated, with both the cultures 
developed nodules on their roots, while the controls had none. The 
organism that was isolated on October 10, 1910, therefore, was the causal 
organism of nodules on Canada field pea plants, and as far as could be 
determined by laboratory methods it was also a pure culture. 

The two cultures of the organism — the one isolated originally, and 
the other isolated from a nodule of a plant grown under sterile conditions 
— were studied in the laboratory with respect to their morphology and 
their physiological activities. An exhaustive study of this phase was not 
undertaken, the study being carried only far enough to show whether 
the two cultures were the same organism. The study consisted in propa- 
gating the two cultures on various media and in comparing and describing 
their cultural and biochemical features. The descriptions were recorded 
on the Descriptive Chart adopted for such use by the Society of American 
Bacteriologists (1907). 

Morphology oj the organism 

Bacillus radicicola of Canada field pea produces no spores when prop- 
agated on the artificial media in the laboratory. In a young culture on 
artificial media, the organism is in the form of small rods about one micron 
long. In this form it is able to multiply by fission, like other bacteria. 
Under certain conditions — for example, with the addition of certain 
nutrients, such as sugar, to the media — some of these small rods develop 
into large cells, which are generally called bacteroids. Some of the bac- 
terids assume the characteristic X and Y forms, the same as are found 
in the nodules. The development of the bacteroids seems to be largely 
a matter of nutrition, and the bacteroids are not a degenerate form, but 
a normal form, of the organism. 

In a culture twenty-four hours old on an agar slope the organism is 
very motile. As the agar-slope culture gets older, fewer and fewer of the 
organisms show motility, until in a culture about two weeks old no motility 



Studies of Bacillus Radicicola of Canada Field Pea 17 

is detected. Motility seems to be influenced by environmental conditions. 
This point has not been studied extensively, beyond the observation that on 
nitrogenous media the motility is lost sooner than on nitrogen-free media. 

Since the generic name of the organism depends on the presence and the 
place of attachment of flagella, and since there has been so much un- 
certainty on this point, an effort was made to demonstrate the number 
and the arrangement of the flagella. Beyerinck (1890) was the first who 
claimed to have isolated the organism in a pure culture. He described 
it as having one polar flagellum, and named it Bacillus radicicola. In 
1905, Moore, agreeing with Beyerinck as to the number of flagella and 
wishing to conform to Migula's classification, changed the generic name 
from Bacillus to Pseudomonas. Edwards and Barlow (1909) found only 
one long, whiplike flagellum, thus agreeing with Beyerinck and Moore. 
De' Rossi (1907) was the first investigator who found the organism of 
Vicia faba to have about eight flagella with a peritrichic arrangement. 
Zipfel (1912) agreed with de' Rossi, stating that the organism has numerous 
flagella. In 1912 Kellerman, using a special method, also succeeded in 
staining the organism of several leguminous plants. He likewise found 
the organism to have several flagella peritrichically arranged. 

In this investigation the following method was used for staining the 
organism of Canada field pea for flagella: 

An agar slope culture twenty hours old, on medium 335 at 24° C, was 
very carefully transferred into 3 cubic centimeters of sterile distilled water 
in a test tube. This was allowed to stand for about four hours at a con- 
stant temperature, without being shaken or disturbed in any way. A 
drop of this infusion was placed on a cover glass and allowed to dry at 
room temperature, and when dry it was stained. 

It was essential that the cover glasses should be clean. To this end 
they were treated with a cleaning solution of potassium bichromate and 
sulfuric acid, washed in water, placed in alcohol, and finally dried with 
a piece of cheesecloth which had been treated with ether in order to get 
rid of any fats. The cover glasses were then placed in a petri dish and 
baked in the oven for three hours at 200° C. If cover glasses are treated 
in this way a large amount of difficulty in the staining of flagella is 
avoided. 

Pitfield's mordant as modified by Muir, and carbol iuchsin, were used 
for staining. The mordant has the following composition: 



18 Martin J. Prucha 



Cubic 
centimeters 



Tannic acid, 10 per cent aqueous solution 10 

Corrosive sublimate, saturated aqueous solution 5 

Alum, saturated aqueous solution 5 

Carbol fuchsin 5 

The mordant was applied for six minutes and the preparation was then 
very thoroughly washed with water. Carbol fuchsin was then applied for 
nine minutes. 

It was found that the organism from Canada field pea has peritrichic 
arrangement of flagella. The largest number of flagella observed was six, 
arising from any part of the organism, and the indications were that the 
organism may have an even larger number. According to Migula's 
classification the organism is Bacillus. 

Cultural and biochemical features 

The surface colonies in a petri dish on agar medium 335 are colorless, 
watery, and very viscous. When ten days old, at 20° C, they are about 
3 millimeters in diameter, although occasionally colonies 10 millimeters 
in diameter may develop. The colonies under the surface are invariably 
of spindle shape. Under the 16-millimeter objective the microscopic 
structure appears granular. 

On agar slope with peptone and beef extract, the growth is watery, 
scanty, and colorless at first; after long standing it becomes brownish. 
In standard gelatin stab the growth becomes brownish. On agar slope 
with medium 335 to which 0.5 per cent of potassium or calcium nitrate 
had been added, the growth becomes opaque and iridescent. In all the 
standard media free from sugar the growth is scanty. In the presence 
of dextrose, saccharose, maltose, or glycerin, much more abundant growth 
takes place. Lactose and galactose increase the growth only slightly, 
while the addition of levulose tends to inhibit it. Two per cent of levulose 
added to media entirely inhibits the growth. Whether this is due to this 
sugar or to some impurity in it was not determined. 

The organism does not produce any indol, hydrogen sulfite, or ammonia. 
It does not reduce nitrates and does not liquefy a 12-per-cent gelatin 
stab at 20° C, but when grown in milk it partly digests the casein without 
curdling the milk. It does not produce any gas from the sugars in fer- 



Studies of Bacillus Radicicola of Canada Field Pea 19 

mentation tubes. From dextrose, maltose, and saccharose it produces a 
slight amount of some acid. Neutral litmus milk becomes alkaline after 
the organism has grown in it for about two weeks. 

The organism of Canada field pea does not have any strikingly charac- 
teristic colony features by which it can be distinguished from other 
microorganisms. The group number of the organism, according to the 
chart of the Society of American Bacteriologists, was found to be 
B. 222.2322033. 

PART II. INFLUENCE OF CERTAIN FACTORS ON NODULE PRODUCTION 

The literature on the general subject of legume inoculation is very 
extensive; yet knowledge concerning the factors that influence nodule 
production is suprisingly meager. For the most part the investigations 
on this point, are incidental and fragmentary. It has been shown by 
various investigators, however, that the nature of the medium in which 
the plant is grown has an influence on the production of nodules. 

Rautenberg and Kiihn (1864) grew Vicia faba in various nutrient 
solutions, and incidentally observed that in the nitrogen -free solution the 
beans developed an abundance of nodules, while in the solutions con- 
taining nitrogen no nodules were produced. 

Hugo de Vries (1877) made a similar observation. He grew red clover 
in nutrient solutions, and the plants developed a large number of nodules 
in the nitrate-free solution but only a few or no nodules in the solution 
containing nitrates. 

Vines (1888-1889) tested the influence of potassium nitrate on nodule 
development on Vicia fabd grown in nutrient solutions and also in the 
soil. His experiments showed that potassium nitrate tends to inhibit 
nodule development, both in soil and water cultures. 

Frank (1889) grew lupines and peas in humus soil and in humus-free 
soil. He found that the plants grown in the humus-free soil developed 
an abundance of nodules, and those grown in the soil rich in humus had 
no nodules. He offered the following explanation for this: " The lupines, 
and also the peas, obtain the same benefit from the nodule fungus as they 
do from the humus. Where humus is present, the plants prefer to obtain 
the nourishment from the humus and no nodules are developed; where 
humus is wanting, however, the nodule fungus infects the plants." 4 

4 Translation from the oiiginal German. 



20 Martin J. Prucha 

Hiltner (1900) showed that the addition of potassium nitrate to the 
nutrient solution in which legumes are grown has an injurious influence 
on nodule development, and he thinks this is due to the fact that the 
formation of bacteroids in the small nodules is hastened by the presence 
of the nitrate. He considers the bacteroids as degenerate and inactive 
forms of the nodule-forming organism. 

A somewhat more extensive investigation of this subject was under- 
taken by Nobbe and Richter (1902). They attempted to determine the 
influence of potassium nitrate and of humous substances on the fixation 
of nitrogen by soy beans. They grew the plants in flowerpots in a rich 
soil and in a poor soil. For rich soil they used garden soil. The poor 
soil was prepared by mixing 4000 grams of sand and 2500 grams of garden 
soil. Potassium nitrate was added to the poor soil in the proportions of 
500 and 1000 milligrams to 0500 grams of the soil. When the plants 
were harvested the total amount of dry substance and of nitrogenous 
matter was determined in each plant. The results of this experiment 
show that the function of the nodules for nitrogen fixation is injured to 
a high degree by the presence of potassium nitrate. The influence of 
humous substances is similar, but not so great. 

The observation made by Frank, by Nobbe and Richter, and by others 
— namety, that soil rich in humous substances has a deleterious effect on 
nodule development — was confirmed also by Moore (1905). He grew 
soy beans in rich nitrogenous soil, in poor clay soil, and in poor sandy 
soil. Very few nodules developed on trie soy beans grown in rich soil, 
while in the poor clay soil and in the poor sandy soil the plants developed 
an abundance of nodules. 

The development of nodules may also be affected by other agencies. 
Gain (1893) attempted to determine the influence of moisture on nodule 
development on- Pimm sativum, two varieties of Lupinus albus, and 
Faba vulgaris. He grew the plants in a field located in a region where 
rain was very scarce during the early part of the summer. One-half of 
the plat planted with each legume was watered artificially, and the other 
half was exposed to drought. Examination of the plants showed that 
the plants watered artificially had five and one-half times as many nodules 
as those not watered. 

Marchal (1901) determined the influence of fifteen different nutritive 
mineral salts on nodule development on peas grown in Sachs' nutrient 



Studies of Bacillus Radictcola of Canada Field Pea 21 

solution. He concluded from his experiments that nodule development 
is inhibited by the addition of the following nutrient salts in the given 
concentrations : 

Alkaline nitrates, concentration 1 to 10,000 
Ammonium salts, concentration 1 to 2,000 
Potassium salts, concentration 1 to 308 3.00 
Sodium salts, concentration 1 to 28§ 

The influence of phosphates was variable, and calcium and magnesium 
salts stimulated nodule development. Marchal was of the opinion that 
the variation of the osmotic pressure, due to the presence of the different 
salts, may be the cause of this phenomenon. 

Moore (1905) states that the addition of 1 per cent of sodium and 
potassium salts often entirely inhibits the formation of nodules, and 
smaller quantities considerably reduce their formation. The addition of 
calcium and magnesium salts, on the other hand, greatly favors nodule 
formation. Moore states further that this is not true with all the legumes, 
since the addition of calcium and magnesium carbonates is injurious 
to the formation of nodules on lupines and other plants adapted to 
acid soil. 

In the following experiments the influence of various factors on nodule 
production has been investigated. The factors studied are light and 
darkness, aeration, moisture, various concentrations of nutrient solutions, 
and a considerable number of chemical substances. 

EXPERIMENT 1 

INFLUENCE of aeration in light and in darkness 

In this experiment Canada field peas were grown in an aqueous soil 
extract. The extract was prepared by taking one part of soil and four 
parts of water, by weight. The mixture was allowed to stand for two 
hours, and the liquid was then decanted and filtered. Twelve Erlen- 
meyer flasks of 300 cubic centimeters capacitj^ were filled, and one pea 
was planted in each flask. Six of the flasks were covered with black paper 
and the other six were exposed to diffused light. Three flasks n each 
of the two series were aerated by passing a current of air through the 
liquid during the entire experiment. All the plants were inoculated with 



22 Martin J. Prucha 

a pure culture of Bacillus radicicola. After twenty-four days the plants 
were examined for nodules. 

Results 

All the plants developed plenty of nodules. The inoculation with the 
pure culture had no apparent effect on the number of nodules. The soil 
extract was not sterilized and apparently had plenty of the organisms. 
The plants whose roots were kept in darkness had a greater abundance 
of nodules than those whose roots were exposed to light. The aeration as 
supplied in this experiment had no stimulative effect on either the number 
or the size of the nodules. 

EXPERIMENT 2 
INFLUENCE OF SOME NUTRIENT SOLUTIONS IN LIGHT AND IN DARKNESS 

Ninety Erlenmeyer flasks of 300 cubic centimeters capacity were 
divided into five series, with eighteen flasks in each series. These flasks 
were filled with the following solutions, respectively: series 1, with medium 
101; series 2, with Crone's solution; series 3, with Pfeffer's solution; series 
4, with tap water; series 5, with soil extract (the same as was used n 
experiment 1). Six flasks from each series, three covered with black 
paper and three not covered, were inoculated with a pure culture of Bacillus 
radicicola. A second group of six flasks from each series were prepared in 
the same manner, but each flask was inoculated with 5 cubic centimeters 
of soil extract. A third group of six flasks from each series were prepared 
in the same manner but were not inoculated. One plant was grown in 
each flask. The water of transpiration was replaced each week. At the 
end of four weeks the plants were examined for nodules. The results are 
given in table 2. 

Results 

Not all the plants that were inoculated developed nodules. A few 
nodules developed on the plants grown in medium 101, in Crone's solution, 
and in Pfeffer's solution. In the soil extract all the plants developed 
nodules, although the plants grew better in Crone's solution and in Pfeffer's 
solution. In the tap water no nodules developed. More nodules 
developed on the roots kept in darkness than on those exposed to the 
light. 



Studies of Bacillus Radicicola of Canada Field Pea 23 
TABLE 2. Influence of Some Nutrient Solutions on Nodule Development 



Culture solution* 






Number of nodules 






Plant 1 


Plant 2 


Plant 3 




( • { 
Inoculated with B. radicicola \ 


Light 


None 


None 


None 




Dark 


None 


None 


None 


Medium 101 


Inoculated with soil extract. j 


Light 


None 


None 


None 


Dark 


None 


None 


Few 




Not inoculated j 


Light 


None 


None 


None 




Dark 


None 


None 


None 




Inoculated with B. radicicola \ 


Light 


None 


None 


None 




Dark 


Few 


Few 


Few 


Crone's solution. . . 


■ Inoculated with soil extract . -1 


Light 


None 


None 


None 


Dark 


Few 


Few 


Few 






Light 


None 


None 


None 






Dark 


None 


None 


None 




Inoculated with B. radicicola < 


Light 


None 


None 


None 




Dark 


None 


None 


Few 


Pfeffer's solution . . 


Inoculated with soil extract . \ 


Light 


None 


Few 


Few 


Dark 


None 


Few 


Few 






Light 


None 


None 


None 






Dark 


None 


None 


None 




Inoculated with B. radicicola I 


Light 


None 


None 


None 




Dark 


None 


None 


None 


Tap water 


Inoculated with soil extract . \ 


Light 


None 


None 


None 


Dark 


None 


None 


None 






Light 


None 


None 


None 






Dark 


None 


None 


None 




Inoculated with B. radicicola \ 

I 


Light 


Present 


Present 


Present 


Soil extract 


Dark 


Present 


Present 


Present 


j 


Light 


Present 


Present 


Present 






Dark 


Present 


Present 


Present 



* See page 11. 



24 Martin J. Prucha 

EXPERIMENT 3 
INFLUENCE OF POTASSIUM NITRATE IN LIGHT AND IN DARKNESS 

In this experiment twelve glass cylinders, each of 5 liters capacity and 
50 centimeters in height, were used. Six of these were filled with Crone's 
full nutrient solution, and the other six were filled with the same solution 
except that potassium chloride was substituted in place of potassium 
nitrate. Five plants were grown in each cylinder. The experiment was 
arranged in the following manner: 

Series 1. Three of the cylinders filled with Crone's full nutrient solution 
were covered with black paper. Two of these were inoculated, and one 
was not inoculated. 

Series 2. The other three cylinders filled with Crone's full nutrient 
solution were treated as was series 1, but were not covered with black 
paper. 

Series 3. Three of the cylinders filled with Crone's solution in which 
potassium nitrate was replaced by potassium chloride, were covered with 
black paper. Two of these were inoculated, and one was not inoculated. 

Series 4. The remaining three cylinders, with the same solution as was 
used for series 3, were treated as was series 3 but were not covered with 
black paper. 

Results 
When the plants were three weeks old, those grown in the solution 
with nitrate looked green and healthy, while those grown in the solution 
without nitrate were turning yellow and the lower leaves were dropping 
off No difference in appearance was observed between the inoculated 
and the uninoculated plants. The uninoculated plants had no nodules; 
those grown in the presence of nitrate and inoculated had one or two 
nodules each ; those grown in nitrate-free solution had about fifteen nodules 
each. Six weeks after planting, the plants grown in nitrate solution had 
thick, green leaves and thick roots, and no more nodules had developed 
on the inoculated plants. The plants grown in nitrate-free solution were 
yellowish except for the upper leaves, which were green; the roots were 
longer and more abundant than on the plants grown in nitrate solution. 
Nodules were abundant, continually developing on the new roots. The 
uninoculated plants in nitrate-free solution had no nodules and were 
practically dead. 



Studies of Bacillus Radicicola of Canada Field Pea 25 

The plants grown in nitrate-free solution with their roots exposed to 
light were slightly shorter than, and did not have quite as many nodules 
as, those grown in the same solution but with their roots kept in darkness. 
In the presence of the nitrate the development of certain green algae 
interfered somewhat with root growth. 

experiment 4 

influence of potassium nitrate and calcium nitrate in pfeffer's 

solution 

The procedure in this experiment was the same as in experiment 3. 
Only one plant was grown in each cylinder. Calcium nitrate was replaced 
by calcium chloride, and potassium nitrate was replaced by potassium 
chloride. The plants were kept until they began to blossom. 

Results 

In the solution with the nitrates, two or three nodules developed on each 
plant within twelve days after inoculation. No more nodules developed 
after that. In the cylinders not covered with black paper, algae developed 
in abundance, and, surrounding the roots, dwarfed the plants. In the 
cylinders covered with black paper, also, some algae developed in time, 
but they were far less abundant. 

In the nitrate-free solution there was an abundant development of 
nodules. The nodules were more numerous on the plants grown in the 
cylinders covered with black paper than on those the roots of which 
were exposed to light. The root system of the plants grown in the nitrate- 
free solution was more developed than that of the plants grown in the 
nitrate solution. 

An interesting point observed in this experiment and in experiment 3 
was that the nodules developed, both in the nitrate solution and in the 
nitrate-free solution, immediately after inoculation. In the nitrate solu- 
tion, however, no further development of nodules took place, while in 
the nitrate-free solution there was a continual development of new nodules 
on the new rootlets as time went on. This.would seem to indicate either 
that the nodule-forming organisms were made inactive by the nature of 
the solution, or that the solution in some way affected the resisting power 
of the plants. 



26 Martin J. Prucha 

EXPERIMENT 5 

INFLUENCE OF VARIOUS CONCENTRATIONS OF PFEFFER's SOLUTION, BOTH 
WITH AND WITHOUT NITRATES 

Wide-mouth bottles of 500 cubic centimeters capacity were used in 
this experiment. They were all covered with black paper. Two series 
were prepared, one with Pfeffer's full nutrient solution and the other 
with Pfeffer's solution in which the nitrates were replaced by the chlorides 
of the same metals. The following concentrations in each series were 
employed, taking the concentration of the full nutrient as 1: &, |, |, \, 
1, 2, 3, 4, and 8. Five plants were grown in each bottle, and all were 
inoculated. The duration of the experiment was three weeks. 

Results 

In the full nutrient solution a few nodules developed in the ^ concen- 
tration. In the other concentrations no nodules appeared within the three 
weeks. 

In the nitrate-free solution nodules developed best in concentration 1. 
In the \ and \ concentrations a few nodules appeared. 

EXPERIMENT 6 

INFLUENCE OF PFEFFER's SOLUTION IN WHICH THE ESSENTIAL ELEMENTS 

WERE ABSENT 

In this experiment the same vessels were used as in experiment 5. 
The solutions were prepared as follows: 

j? , , • • ., f Ca(N0 3 )2 was replaced by CaCl 2 

t or solution minus nitrogen \ T „^ , ,, T ;L„ 

{ KN0 3 was replaced by KC1 

fKN0 3 was replaced by NaN0 3 
For solution minus potassium <j KH 2 P0 4 was replaced by NaH 2 P0 4 

[ KC1 was replaced by NaCl 
For solution minus phosphorus, KH 2 P0 4 was replaced by Jfrilt s PCX >< <% 
For solution minus sulfur, MgS0 4 was replaced by MgCl 2 
For solution minus magnesium, MgS0 4 was replaced by MgGl 2 Xe^SO^ 
For solution minus iron, FeCl 3 was replaced by NaCl 

One plant was grown in each bottle. The plants were examined three 
weeks after planting. 



Studies of Bacillus Radicicola of Canada Field Pea 27 

Results 
Nodules developed only in the nitrate-free solution. The plants in 
most of the solutions in this experiment and in experiment 5 did not grow 
well and normally. It is possible that slightly different results might 
have been obtained had the length of time of the experiment been extended. 
It was observed, however, in these and in the other experiments, that the 
number and the size of the nodules on a plant are influenced by the rate 
and the amount of growth of the plant. In other words, any disturbing 
factor in the normal functions of a plant tends to hinder the development 
of nodules. 

EXPERIMENT 7 

INFLUENCE of moisture 
In experiments 7, 8, and 9, the plants were grown in glass tumblers. 
In each tumbler was placed 300 grams of air-dry sandy soil containing less 
than 0.5 per cent of moisture. The following percentages of moisture 
were used: 5, 10, 15, 20, 25, 30, 40, 50, and 60, the percentage being based 
on the air-dry soil. Three tumblers were used for each percentage of 
moisture, and five plants were grown in each tumbler. The plants were 
kept in the greenhouse. They were watered every other day, the neces- 
sary amount of water to be added being determined by weighing. The 
soil is naturally well inoculated with the Canada field pea organism, but 
in addition to this each tumbler was inoculated with a pure culture of the 
organism. The duration of the experiment was four weeks. 

Results 
The best growth took place in 15, 20, and 25 per cent of moisture. 
In 5 and 10 per cent of moisture the plants grew very slowly, while in 50 
and 60 per cent the roots rotted. Nodules were present on all the plants. 
The number of the nodules on each plant increased with the percentage 
of moisture up to 40 per cent. These results agree with those of Gain 
(1893), who found that a larger number of nodules develop when the 
plants are abundantly watered. 

EXPERIMENT 8 
INFLUENCE OF CERTAIN SUBSTANCES IN VARYING QUANTITIES 

The same soil and the same kind of vessels were used in this experiment 
as in experiment 7. Three hundred grams of the air-dry soil was in- 



28 



Martin J. Prucha 



troduced into each glass tumbler; the substance to be tested was dissolved 
in the proper quantity of water, and this was added to each tumbler. 
Five plants were grown in each tumbler, and all the cultures were made 
in triplicate and were inoculated. The plants were allowed to grow for 
four weeks. The kind and the amount of the substance added, together 
with the results, are given in table 3: 



TABLE 3. 



Influence on Nodule Development of Certain Substances in Varying 
Quantities 



Substance used 



Quantity 

added to 

300 grams 

of soil 

(grams) 



Condition of plants 



Nodule development 



KNO s 



0.25 
0.50 
1.00 
2.00 



Good growth 
Poor growth . 
No growth . . 
No growth . . 

Good growth 
Poor growth . 
No growth. . 
No growth . . 

Good growth 
Good growth 
Good growth 
Good growth 

Good growth 
Good growth 
Good growth 
Good growth 

Good growth 
Good growth 
Good growth 
Good growth 

No growth . . . 
No growth . . . 
No growth. . . 
No growth . . . 

No growth . . . 
No growth . . . 
No growth . . . 
No growth . . . 



No nodules 
No nodules 
No nodules 
No nodules 



Ca(N0 3 ) 2 . 



0.25 
0.50 
1.00 
2.00 



Few nodules 
No nodules 
No nodules 
No nodules 



MgSO, 



KH2PO4. 



CaCO,. 



NII4CI. 



0.25 
0.50 
1.00 
2.00 



0.25 
0.50 
1.00 
2.00 



0.25 
0.50 
1.00 
2.00 



0.25 
0.50 
1.00 
2.00 



FeCl, 



0.25 
0.50 
1.00 
2.00 



Nodules abundant 
Nodules abundant 
Nodules abundant 
Nodules abundant 



Nodules abundant 
Nodules abundant 
Nodules abundant 
Nodules abundant 



Nodules very abundant 
Nodules very abundant 
Nodules very abundant 
Nodules very abundant 



No nodules 
No nodules 
No nodules 
No nodules 



No nodules 
No nodules 
No nodules 
No nodules 



Studies of Bacillus Radicicola of Canada Field Pea 



29 



TABLE 3 (concluded) 



Substance used 


Quantity 

added to 

300 grams 

of soil 

(grams) 


Condition of plants 


Nodule development 


r 


0.25 
0.50 
1.00 
2.00 


Fair growth 


Few nodules 


Witte's peptone \ 


Poor growth 

Poor growth 

Poor growth 


No nodules 
No nodules 
No nodules 


Cane sugar • 


0.25 
0.50 
1.00 
2.00 
4.00 
8.00 
16.00 


Good growth 

Good growth 

Good growth 

Good growth 

Poor growth 

Poor growth 

Very poor growth. . . . 


Nodules present 
Nodules present 
Nodules present 
Nodules present 
Nodules present 
No nodules 
No nodules 


Controls 


Nothing 
added 


Good growth 


Nodules present 



Results 
The addition of MgS0 4 , KH 2 P0 4 , and CaC0 3 in the concentrations 
used in the experiment had a beneficial effect on the development of nod- 
ules. Cane sugar at low concentrations had apparently no effect. At 
the concentrations of 4, 8, and 16 grams of sugar in 300 grams of soil, 
cane sugar was injurious, probably due to fermentation products and to 
stimulation of the development of microorganisms injurious to the plants 
and also to the development of nodules. The addition of NH 4 C1 and 
FeCl 3 completely inhibited the growth of the plants. In the case of 
KN0 3 and Ca(N0 3 ) 2 the concentration of \ gram in 300 grams of soil had 
a beneficial effect on the growth of the plant, but an injurious effect on 
the development of nodules. A few nodules developed in the presence of 
Ca(N0 3 )2, but none in the presence of KN0 3 . The higher concentrations 
of Ca(N0 3 ) 2 and KN0 3 inhibited nodule development and also caused 
injury to the plants. 

EXPERIMENT 9 

INFLUENCE of certain additional substances 
The method used in this experiment was the same as in experiment 8. 
Several additional chemicals were tested. In examining the plants, the 



30 



Martin J. Prucha 



number of nodules was counted and both the total number of nodules 
and the average per plant for each concentration of the substance were 
calculated. The results follow in table 4: 



TABLE 4. Influence of Certain Additional Substances on Nodule Development 



Chemical used 


Quantity 

added to 

300 grams 

of soil 

(grams) 


Number 
of plants 


Condition 
of plants 


Number 

of 

small 

nodules 


Number 

of 

large 

nodules 


m . i i Number 
1 otal | , 

number , , 
, nodules 

nodules i pknt 


Ca(N0 3 )2 


0.25 
0.50 
1.00 
2.00 


10 
8 




Small 

Small 

Plants killed 
Plants killed 


6 




6 



0.6 




Tannic acid — j 


0.25 
0.50 
1.00 

2.00 


11 

8 

12 

13 


Good 

Good 

Good 

Good 


40 

50 

17 

100 


138 
41 
50 
20 


178 
91 
67 

120 


16.2 

11.4 

5.6 

9.2 


KH 2 PC-4 


0.25 
0.50 
1.00 

2.00 


6 
7 
4 
4 


Good 

Good 

Small 

Small 


10 
50 
30 
16 


50 

58 

10 

5 


60 

108 

40 

21 


10.0 

15.4 

10.0 

5.3 


f 
MgSO« | 


0.25 

0.50 

1.00 

' 2.00 


14 

11 
12 
13 


Good 

Good 

Good 

Good 


46 
26 
60 
24 


100 
50 
41 
32 


146 
76 

101 
56 


10.4 
6.9 
8.4 
4.3 


KC1 | 


0.25 
0.50 
1.00 
2.00 


7 





Small 

Plants killed 
Plants killed 
Plants killed 


34 


12 


46 


6.6 


KNO-3 


0.25 
0.50 
1.00 
2.00 


14 

14 

3 




Good 

Small 

Plants killed 
Plants killed 


15 





15 


1.1 


NH,C1 | 


0.25 
0.50 
1.00 
2.00 


12 

2 




Good 

Small 

Plants killed 
Plants killed 


















( 
Witte's peptone \ 


0.25 
0.50 
1.00 
2.00 


11 

11 

9 

1 


Good 

Good 

Small 

Very small. . 


21 

20 

4 


8 
2 



29 

22 

4 


2.6 
2.0 
0.4 



Studies of Bacillus Radicicola of Canada Field Pea 31 

TABLE 4 (concluded) 



Chemical used 


Quantity 

added to 

300 grams 

of soil 

(grams) 


Number 
of plants 


Condition 
of plants 


Number 

of 

small 

nodules 


Number 

of 

large 

nodules 


Total 
number 

of 
nodules 


Number 

of 

nodules 

per 

plant 


KOH J 


25 
0.50 
1.00 
2.00 


12 

10 

3 




Good 

Good 

Small 

Plants killed 


35 

10 
11 


50 
45 
25 


85 
55 
36 


7.1 

5.5 

12.0 


Fe(N0 3 ) 3 


0.25 
0.50 
1.00 
2.00 


11 

14 

6 




Good 

Good 

Small 


4 
1 








4 
1 



0.4 
0.1 









Ca(H 2 PO0 2 .... | 


0.25 
0.50 
1.00 
2.00 


8 
14 
12 

5 


Good 

Good 

Good 

Small 


6 
90 
54 

8 


45 
94 
60 
11 


51 
184 
114 

19 


6.4 

13.1 

9.5 

3.8 


CaSO< 


0.25 
0.50 
1.00 
2.00 


11 
10 
14 
10 


Good 

Good 

Good 

Good 


22 
15 

57 
21 


65 
60 
50 
45 


87 

75 

107 

66 


7.9 
7.5 
7.4 
6.6 


FeCh 


25 
0.50 
1.00 
2.00 








Plants killed 
Plants killed 
Plants killed 
Plants killed 










Starch < 


1.00 
2.00 
4.00 


12 
13 

8 


Good 

Good 

Good 


40 
59 
18 


82 
40 
42 


122 
99 
60 


10.2 
7.6 
7.5 


Controls j 




18 
14 


Good 

Good 






113 
107 


6.3 
7.6 



Results 
The results of experiment 9 are similar to those of experiment 8. The 
following chemicals added to the soil at the concentrations used in the 
experiment were injurious to the plants and tended to inhibit the de- 
velopment of nodules: Ca(N0 3 ) 2 , KC1, KN0 3 , NH 4 C1, Witte's peptone, 
Fe(N0 3 ) 3 , and FeCl 3 . On the other hand, tannic acid, KH 2 P0 4 , MgS0 4 , 
KOH, Ca(H 2 P0 4 )2, CaS0 4 , and starch exerted a beneficial influence on 
nodule development and appeared to have no injurious effect on the plants. 



32 Martin J. Prucha 

GENERAL DISCUSSION OF RESULTS OF EXPERIMENTS 

The nine experiments reported in Part II; on the factors affecting the 
development of nodules on Canada field peas, are not extensive enough 
to allow any broad and general deductions, but the results point to several 
conclusions. Nodules develop readily on Canada field peas in nutrient 
solutions, provided the proper nutrient salts are added. Varying the 
concentration appears to have a marked influence on nodule development. 
Aeration has no appreciable effect. Nodules developed on the long roots at 
as great a depth as 30 centimeters below the surface of the nutrient solu- 
tion. If air is essential for the development of nodules, enough of it was 
dissolved in the nutrient solution under the conditions in the experiments. 
The presence of nitrates in the nutrient solution or in the soil tends to 
inhibit the development of nodules; the reason for this is not known. 
If the plants are grown in the presence of nitrates for about a week and 
then inoculated, a few nodules will develop, but no further development 
of nodules takes place; in water cultures without nitrates and inoculated, 
a continuous nodule development takes place as long as the roots 
grow. 

The chemical composition of the various soils used for agricultural 
purposes differs. What influence this has on the various groups of the 
nodule-forming organism and on nodule development has never been 
extensively investigated. The results of the foregoing experiments em- 
phasize its importance. The limited distribution of the different groups 
of the nodule-forming organism in some soils, the failures in inoculations, 
and the difficulty in growing certain legumes, may be explained in certain 
cases as being due to the composition of the soil. It is known that the 
addition of lime to certain soils has a beneficial effect on nodule develop- 
ment and on the growth of some legumes. It is highly probable that 
the addition of other substances to the soil may be beneficial to other 
legumes. 

PART III. INFLUENCE OF VARIOUS MEDIA ON THE INFECTING POWER 
AND THE VITALITY OF BACILLUS RADICICOLA 

As indicated previously, and again further developed in the discussion 
of this subject, the view has been maintained that the infecting power, 
or " virulence," of the nodule-forming organism may be impaired by 
cultivating it on certain media. In order to determine whether or not 



Studies of Bacillus Radio cola of Canada Field Pea 33 

such is the case, and also to determine the media most favorable for 
maintaining the vitality of the organism, the following experiments were 
made. 

The term virulence has been used by previous investigators to mean 
the ability of the organism to penetrate the root and produce nodules. 
Since the term virulence in this connection, as also suggested by Edwards 
(in Marshall's Microbiologic), does not correctly apply to the legume 
bacteria, the term infecting power will be used throughout this paper. 

Beyerinck (1890) was the first to isolate a pure culture of an organism 
from a nodule. Prazmowski (1890), Frank (1889), and Nobbe and others 
(1891), stimulated by Beyerinck's success, were also able to obtain pure 
cultures from the nodules of various legumes, and to produce nodules by 
inoculating the plants with the pure cultures. The experiments on the 
inoculation of legumes by pure cultures at once raised a question as to 
the classification of the nodule-forming organism, which question is in- 
timately connected with the subject of the infecting power of the organism 
and the resistance of the plants. Do all the different organisms from 
the various species of legumes belong to several species, or do they belong 
only to one species but to several races or varieties? Can the organism 
from one species of legumes cause nodules on a different species of legumes? 
Is the relation between the legume and the organism a case of symbiosis 
or a case of parasitism? Does the organism have the biological or physio- 
logical character called virulence as understood by pathologists, and can 
this be altered or destroyed without injuring or destroying the other 
physiological activities? Do the host plants have a resistance in a patho- 
logical sense, and can this resistance be altered by the environmental 
factors without altering the morphology, the structure of the tissues, 
and the physiological activities of the plants? Is the resistance against 
the entrance of the organism into the root tissues different from the 
resistance against the development of the organism inside the root tissues? 
These and similar questions formed the foundation of the numerous 
investigations that were undertaken subsequently to the isolation of the 
pure culture of the nodule-forming organism, and nodule production by 
pure cultures. 

Even before the isolation of the nodule-forming organism by Beyerinck, 
it was observed by Hellriegel (1886) that when peas, vetch, -beans, clover, 
serradella, and lupines were inoculated with an infusion from the same 



34 Martin J. Prucha 

soil, all the plants developed nodules except the serradella and the lupine. 
From this Hellriegel inferred that important differences must exist between 
the nodule bacteria of the different legumes. 

Beyerinck (1888) was of the opinion that the nodule-forming organisms 
of the legumes belonged to one species, but that there were several groups 
and in each group a number of varieties. From the results of his sub- 
sequent investigation (1890) he was forced to change his former opinion. 
He considered the organism of Ornithopus and that of Vicia to be two 
distinct species. 

Frank's investigations (1899) led him to believe that there was only 
one species among the nodule-forming organisms. 

Salfeld (1888) grew peas and horse beans in " Hochmoorboden," and 
inoculated one part of them with sandy soil in which peas were grown 
and the other part with sandy soil in which lupines were grown. Both 
the peas and the horse beans inoculated with the pea soil developed 
nodules, while those inoculated with the lupine soil were free from nodules. 

Laurent (1901) could produce nodules on dwarf peas by inoculating 
them with material from nodules of thirty different leguminous species, 
but he claimed that the number, size, and appearance of the nodules 
was influenced by the inoculating material of the different sources. 

Kirchner (1896) grew about one hundred different species of legumes 
in the Hohenheimer botanical garden. He observed that all the different 
species of legumes developed nodules in the garden soil except the soy 
beans, although these had been grown in the garden for ten years. The 
soy beans did not produce nodules until they were inoculated with soil 
on which Japanese soy beans had been grown. 

The investigations of Maze (1898) led him to divide the nodule-forming 
organisms into two groups — those adapted to a neutral or an alkaline 
soil, and those adapted to an acid soil; the former infecting the plants 
that favor neutral and alkaline soil, and the latter infecting the plants 
that favor acid soil. 

Nobbe, Schmid, Hiltner, and Hotter (1891) undertook a very extensive 
series of investigations on the general subject of nitrogen assimilation by 
leguminous plants. Much of the present information on this subject is 
due to these men, especially to Hiltner and Nobbe. They showed that 
the only way to study the relations between the nodule-forming organisms 
of the different legumes and the different species and varieties of legumes 



Studies of Bacillus Radicicola of Canada Field Pea 35 

was to use pure cultures of the organisms for inoculation purposes, and 
not die soil infusions as was done by a number of previous investigators. 

In a subsequent paper, Nobbe, Hiltner and Schmid (1895) arrived at 
the following important conclusions on the relation of nodule-forming 
organisms to the different species of legumes: 

" The infecting power of the nodule bacteria of the various groups 
and species of legumes cannot be differentiated absolutely, but only in 
degree. The pure cultures from nodules of different species of legumes 
do not represent different species, but only different forms. We have 
not the least doubt that all the nodule bacteria of the different legumes 
we have studied, even those of Mimosae, are one species, all belonging 
to Bacillus radicicola of Beyerinck. These bacteria, however, are 
influenced by the plants in whose roots they live to such a degree that 
their descendants are able to infect readily only that species of legumes 
to which the former host plant belonged, at the same time losing partly 
or completely the power to infect other species of legumes. When the 
legume is grown in a suitable soil, nodules will develop on the roots 
only when either those nodule bacteria are present which have lived 
previously on that legume species, or when the neutral nodule bacteria 
are present. The latter will be found in the soil where legumes have never 
been grown or where they have not been grown for a long time. If one 
legume is preponderantly grown in a soil, most of the neutral bacteria 
become influenced by this legume, and when a different legume is planted 
which is not closely related to the former no nodules will be formed, or 
only very few and faulty ones, and these will appear so late that they 
will be of very little value to the plants." 5 

By means of extensive experiments (Nobbe and Hiltner, 1896) it has 
been demonstrated that effective inoculation is obtained only when the 
plants are- inoculated with bacteria from the nodules of the same species 
of legumes. 

Moore (1905) conducted extensive cross-inoculation experiments, and 
maintains that "it is possible to cause the formation of nodules upon 
practically all legumes, no matter what was the source of the original 
organisms, provided they were cultivated for some time upon a synthetic 
nitrogen- free medium." He states further: "It is undoubtedly true that 
the long adaptation of the bacteria to the special conditions obtaining 

5 Translation from the original German. 



36 Martin J. Prucha 

in a particular species of legume enables such organisms to produce more 
abundant nodules in a shorter length of time than bacteria isolated from 
some other legume and grown upon nitrogen-free media. While this is of 
considerable practical importance, and will probably always make it neces- i 
sary to distribute the specific organism for the specific crop, it does not in 
any way indicate that the bacteria found in the nodules of beans, peas, 
clovers, etc., are separate species. The most that can be maintained is] 
that there is a slight physiological difference due to the long association 
with a plant of a peculiar reaction which enables the bacteria more easily 
to penetrate the host upon which they have been accustomed to grow. 
These slight racial characteristics can readily be broken down by culti- 
vation in the laboratory, and it is entirely possible to secure a universal 
organism capable of producing a limited number of nodules upon all the 
legumes which now possess these growths." 

Hopkins (1904) found that the organism from sweet clover readily 
inoculates alfalfa. 

Nobbe and Hiltner (1900) undertook to train the nodule- forming 
organism of peas and that of beans so that the former may cause nodules 
on beans and the latter on peas. They succeeded in doing this, and 
drew the following conclusions: 

1. The nodule-forming organism from peas can be trained to produce 
nodules .on beans, and that from beans to produce nodules on peas. 

2. Although some nodules are produced in both cases, the organisms 
do not assimilate any nitrogen at first. 

3. If the pea organism that caused nodules on beans is isolated and 
beans are inoculated with it a second time, the organism then infects the 
beans more readily than at the first inoculation and its power to assimilate 
nitrogen is increased. The organism of beans behaves in the same manner 
when made to infect peas. 

Kellerman (1912) reports that Mr. Leonard has succeeded in securing 
abundant inoculation on soy beans, lupines, and alfalfa from an organism 
of a culture originally isolated from the alfalfa nodule and kept on an 
artificial medium in the laboratory for about six years. Kellerman, 
therefore, is of the opinion that the nodule-forming organisms of all the 
Leguminosee should be considered as a single species. 

The evidence from the investigations mentioned above points to two 
conclusions: (1) that, with some exceptions, the nodule-forming organism 



Studies of Bacillus Radigtcola of Canada Field Pea 37 

from one legume does not cause nodules on another legume; (2) that 
the organism from one legume may be trained to cause nodules on 
any other legume. The evidence for the latter conclusion, however, is 
not final. 

About 1895 a German company placed nitragin on the market — a pure 
culture of the nodule-forming organism for inoculation purposes. The 
cultures were propagated on gelatin and their preparation was based 
on the results of the investigations of Nobbe and Hiltner. These cultures 
were extensively tested both in Germany and in other countries, and, 
as judged by the reports of these tests, the cultures proved only partially 
successful. As a result of these adverse reports on nitragin, Nobbe and 
Hiltner (1899 a) undertook to ascertain the cause of the low efficiency 
of their cultures. They had already shown that the nodule-forming 
organism can be trained to infect other legume species than that of its 
host plant, when they trained the organism from peas to produce nodules 
on beans and that from beans to cause nodules on peas. They went a 
step further and demonstrated that the infecting power of the organisms 
can be altered in degree. They stated that the propagation of the 
organism on artificial media increases or diminishes the vitality, and 
that in general nitrogenous media are injurious to the vitality of the 
organism. 

Moore (1905) also reports that as a result of numerous trials it has 
been found that, although the bacteria increase most rapidly on a medium 
rich in nitrogen, the resulting growth is usually of very much reduced 
vitality, and when put into the soil these organisms have lost the ability 
to break up into the minute forms necessary in order to penetrate the 
root hairs. 

In a further study of this subject, Hiltner (1900) was led to believe that 
this variableness in the infecting power of the nodule-forming organism 
is the determining factor of the number and size of the nodules on every 
plant when grown under otherwise favorable conditions. He took some 
older plants that already had nodules on their roots, and placed them 
in a nutrient solution without any nitrogen. Repeated inoculation 
with its own organism did not produce any nodules on the new rootlets. 
When fall came, and the leaves began to turn yellow and drop, and the 
organisms in the nodules became weaker than those in the solution, 
nodules began to develop on the rootlets. When Hiltner took older 



38 Martin J. Prucha 

plants that had no nodules on their roots and placed them in a similar 
solution to that used with the other plants, an immediate development 
of nodules took place on the new rootlets. From this and other experi- 
ments, Hiltner concluded that " the active nodules produce immunity 
in the plant against the bacteria that possess the same or a lower degree 
of infecting power than those already living in the nodules of that plant. 
Only bacteria of higher infecting power are able to enter the root 
tissue." 15 

- Suchting (1904), believing that Hiltner's theory of the infecting power 
of the organism and its relation to nodule development was not sound, 
undertook a series of interesting experiments on this subject, as well 
as an elaborate discussion of Hiltner's theory and of his own theory. 
In his experiments Suchting attempted to ascertain three points: (1) Have 
the organisms that produce the first nodules on the plant less infecting 
power than those that produce nodules on the same plant subsequently? 
(2) Does the symbiosis with the plant influence the infecting power of 
the organism? (3) Does the artificial medium influence the infecting 
power of the organism? 

From his experiments Suchting drew the following conclusions: 

1. The infecting power of the bacteria is not proportional to the age of 
the nodule. 

2. The passage of the bacteria through the host plant does increase 
their infecting power. Their infecting power does not vary at the different 
stages of the plant's vegetative period, and the feeding of the plant by 
potassium nitrate is injurious to the bacteria in the nodules. 

3. When propagated on artificial media the lupine bacteria lose their 
infecting power on some media and may exist in a so-called pseudo form. 
On neutral media the bacteria retain their infecting power better than on 
acid media. The horse-bean bacteria do not behave in the same manner, 
but keep their infecting power for a long time on suitable media. 

Lewis and Nicholson (1905), on the other hand, found by their experi- 
ments that " It seems that the presence or absence of nitrogen in the 
culture media is not the determining factor in maintaining the activity 
of the germ. Cultivation in the presence of the amount of nitrogen 
usually present in bouillon with from two to five per cent of cane sugar 

•Translation from ths original German. 



Studies of Bacillus Radicicola of Canada Field Pea 39 

or glucose, preferably the former, has given best results in a 1 of the work 
connected with the experiment." 

In the following pages data are presented on experiments conducted 
through several years in an attempt to alter the " virulence " — that 
is, the infecting power — of the organism. In experiments 10, 12, and 13, 
Bacillus radicicola of Canada field pea was used; in experiment 11 that 
of alfalfa was used. The organisms were propagated and kept on various 
media. Their infecting power was tested and measured by the nodule 
development in plants grown in a sterilized sandy soil. 

EXPERIMENT 10 

INFLUENCE of clay, loam, sand, and carborundum 
In this experiment the organism was grown on clay, sandy loam, sandy 
soil, fine quartz sand, coarse quartz sand, and carborundum. One hundred 
grams of each substance, air-dried, was introduced into Erlenmeyer flasks 
of 300 cubic centimeters capacity. After sterilization the media were 
heavily seeded with B. radicicola. This was accomplished by introducing 
into each flask the growth of B. radicicola from one agar slope, along 
with the necessary quantity of water. The amount of moisture added 
to each medium was about five per cent less than its capacity for holding 
water. 

Two series of flasks were prepared. In series 1 the media, as soon 
as seeded with the organism, were spread on sterile paper and allowed 
to dry at room temperature. Th? time required for their complete drying 
was about six hours. In series 2 the media were left in the flasks, plugged 
with cotton, and allowed to stand in the laboratory. Drying of the 
media in this series was very gradual. The infecting power of the organism 
in these cultures was tested by inoculating plants. For this purpose 
Canada field peas were grown in sterilized soil in flowerpots, and were 
inoculated with the respective cultures at the time of seeding. Inoculation 
was accomplished by scattering one gram of the inoculating material 
over the soil in the flowerpots. The first test was made when the cultures 
were ten days old and the second test when the cultures were forty- 
six days old. When the plants were three weeks old they were 
examined for presence of nodules. The results are presented in tables 
5 and 6: 



40 Martin J. Prucha 

TABLE 5. Results of First Inoculation Test. The Cultures Were Ten Days Old 



Plants inoculated with 



Number 

of 

plants 



Number 

of 

plants 

with 

nodules 



Total 
number 

of 
nodules 



Number 

of 

nodules 

per plant 



Clay 

Sandy loam 

Sandy soil 

Fine quartz sand . . 
Coarse quartz sand 
Carborundum 

Clay 

Sandy loam 

Sandy soil 

Fine quartz sand . . 
Coarse quartz sand 

Carborundum 

Agar slope culture. 
Controls 



17 
23 
14 
15 
19 
11 



4 
13 
7 
3 

2 



31 

81 

45 
9 


25 



1(1 
LI 
is 
13 
18 
14 
I'.i 
L3 



51 

'.»() 
(17 
92 
'.it 
23 
90 
15 



5.1 
6.0 
3.7 
7.1 
5.2 
1.6 
4.7 
1.2 



TABLE 6. 



Results of Second Inoculation Test. 
Days Old 



The Cultures Were Forty-six 



Plants inoculated with 



Number 

of 
plants 



Number 

of 

plants 

with 

nodules 



Total 
number 

of 
nodules 



Number 
of 

nodules 
per plant 



Clay 

Sandy loam 

Sandy soil 

Fine quartz sand . . 
Coarse quartz sand 
Carborundum 

Clay 

Sandy loam 

Sandy soil 

Fine quartz sand . . 
Coarse quartz sand 

Carborundum 

Controls 



5 
12 
12 

10 

4 

11 



3 
4 

14 

8 

7 

10 

36 



3 
3 

14 
7 
6 
4 

13 



53 
79 

151 
58 
41 

113 



20 

45 

188 

190 

78 

32 

120 



10.6 
6.6 

12.6 
5.8 

10.3 

10.3 



6.7 
11.3 
13.4 
23.8 
11.1 
3.2 
3.3 



Studies of Bacillus Radicicola of Canada Field Pea 41 

Results 

In both tests there was a certain amount of infection due to other 
sources than the inoculating materials. In the first test three of the 
thirteen plants used as controls developed nodules, while in the second 
test thirteen of the thirty-six control plants developed nodules. It was 
noticed, however, that, as a rule, if contamination took place subsequently 
to the inoculation and the plants were examined within four weeks after 
planting, the nodules due to the contamination were small and developed 
on the lateral roots near the surface of the soil, whereas nodules resulting 
from inoculation always appeared first on the taproot and were larger. 
Nevertheless, the results as shown in the two tables do not allow any 
clear-cut deductions. The plants of the first test (table 5) were examined 
three weeks after planting; the plants of the second test (table 6) were 
kept for four weeks, which probably accounts for the larger number of 
nodules on those plants. 

It appears certain that B. radicicola remained alive and retained its 
infecting power in practically all the substances for forty-six days. Car- 
borundum gave the poorest results. The plants inoculated with this 
substance developed only small nodules, mostly on the side roots — a fact 
pointing to subsequent infection. As regards the two series, much better 
inoculation was obtained from series 2 in both tests. In the first test 
99 plants were inoculated with the cultures of series 1. These plants 
produced 191 nodules, which is an average of 1.9 nodules per plant. The 
88 plants inoculated with the cultures from series 2 produced 417 nodules, 
which is an average of 4 7 nodules per plant. From similar calculations 
for the second test, it is found that the average number of nodules per 
plant was 9.2 in series 1, and 12 in series 2. In both tests the plants 
inoculated with the cultures of series 2 produced more nodules than 
those inoculated with the cultures of series 1. The drying of the substances 
in series 1 either reduced the infecting power of B. radicicola, or reduced 
the number of the organisms, or had both results. 

experiment 11 
influence of hydrochloric acid, sodium hydroxide, and 
cane sugar, in varying concentrations 
In this experiment B. radicicola of alfalfa was isolated and identified 
according to the procedure described in Part I of this paper. The 



42 



Martin J. Prucha 



organism was propagated on media 334, 335, and 337 (page 11), to which 
were added various amounts of hydrochloric acid (HO) and sodium 
hydroxide (NaOH). Cane sugar was added in various amounts to 
medium 334. 

Ten cubic centimeters of the media were introduced into each test 
tube and sterilized. While the agar was still melted the various additions 
were made to the tubes, and, after thorough mixing, the tubes were 
sloped. 

All cultures were made in duplicate. At the end of three weeks stain 
preparations were made from each slope for morphological study, carbol 
fuchsin being used for staining. The media employed and the results 
of the morphological study are given in table 7: 



TABLE 7. Morphological Variation of B. radicicola on the Different Media 




+ 





+ 


(1 


+ 





+ 


1 


+ 


1 


+ 


2 


+ 


3 


+ 


(I 


+ 





+ 


1 


+ 


1 


+ 


2 


+ 


3 


+ 





+ 





+ 


1 


+ 


1 


+ 


2 


+ 


3 


+ 


4 


+ 


5 


+ 


6 


■f 


8 



+ 10 



1 cc. N/1 HC1 
5 cc. N/1 HC1 
Occ. N/1 HC1 
5 cc. N/1 HC1 
cc. N/1 HC1 

cc. N/1 HC1 

1 cc. N/1 NaOH 
5 cc. N/1 NaOH 
cc. N/1 NaOH 
5 cc. N/1 NaOH 
cc. N/1 Na')H 

cc. N/1 NaOH 

1 per cent cane sugar 
5 per cent cane sugar 
per cent cane sugar 
5 per cent cane sugar 
per cent cane sugar 
per cent cane sugar 
per cent cane sugar 
per cent cane sugar 
per cent cane sugar 
per cent cane sugar 
per cent cane sugar. 



Small, short cells 

Short rods, slightly stained 

Short rods, slightly stained 

Short rods, slightly stained 

Short rods, slightly stained 

Short rods, slightly stained 

Short rods, slightly stained 

Short rods, well stained 

Short rods, well stained 

Short rods, well stained 

Short rods, well stained 

Short rods, well stained, few small bacteroids 
Short rods, well stained, few small bacteroids 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids present 

Cells vary in size, bacteroids more abundant 
Cells vary in size, bacteroids more abundant 
Cells vary in size, bacteroids more abundant 
Cells vary in size, bacteroids very abundant 
Cells vary in size, bacteroids very abundant 



Fair 

Fair 

Poor 

None 

None 

None 

None 

Good 

Good 

Poor 

Poor 

Very poor 

Doubtful 

Good 

Good 

Good 

Good 

Good 

Good 

Good 

Good 

Fair 

Fair 

Poor 



Studies of Bacillus Radicicola of Canada Field Pea 43 

TABLE 7 (concluded) 



Medium 



Morphological appearance 



Multiplication 



Medium 335, 10 cc. 

+ 

+ 0.1 cc. N/1 HC1.. 
+ 0.5cc. N/1 HC1. . 
+ 1.0 cc. N/1 HC1. . 
+ 1.5 cc. N/1 HC1.. 
+ 2.0cc. N/1 HC1.. 
+ 3.0cc. N/1 HC1. . 
+ 0.1 cc. N/1 NaOH. 
+ 0.5cc. N/1 NaOH 
+ 1.0 cc. N/1 NaOH. 
+ 1.5 cc. N/1 NaOH 
+ 2.0cc. N/1 NaOH 
+ 3.0cc. N/1 NaOH 

Medium 337, 10 cc. 

+ , 

+ 1 cc. N/1 HC1. . 
+ 0.5cc. N/1 HC1. . 
+ 1.0 cc. N/1 HC1.. 
+ 1.5 cc. N/1 HC1.. 
+ 2 Occ. N/1 HC1.. 
+ 3 Occ. N/1 HC1. . 
+ 0.1 cc. N/1 NaOH 
+ 0.5cc. N/1 NaOH 
+ 1.0 cc. N/1 NaOH 

+ 1.5 cc. N/1 NaOH 

+ 2.0cc. N/1 NaOH 

+ 3 Occ. N/1 NaOH 



Variation in size and shape, few bacteroids. . 
Variation in size and shape, few bacteroids. 

Very few rods 

No organisms visible 

No organisms visible 

No organisms visible 

No organisms visible 

Variation in size and shape, few bacteroids. . 
Variation in size and shape, few bacteroids. . 
Greater variation, more bacteroids than in . 5 
Greater variation, more bacteroids than in 5 
Greater variation, more bacteroids than in . 5 
Cells stained deeper than others, bacteroids 
present 



Good 

Good 

Poor 

Doubtful 

None 

None 

None 

Very good 

Very good 

Good 

Good 

Poor 

Doubtful 



Pronounced variation in shape and size of cells. 

Small cells, irregular shape and size 

Very slender cells, irregular shape and size. . . . 

Extremely small cells 

Extremely small cells 

Extremely small cells 

No organisms visible 

Short, irregular cells 

Short, irregular cells 

Shape and size extremely varied, many bac- 
teroids 

Large cells, varying in shape and size, many 
bacteroids 

Large cells, varying in shape and size, many 
bacteroids 

Very large cells, varying in shape and size, 
many bacteroids 



Good 

Good 

Poor 

Very poor 

Doubtful 

None 

None 

Abundant 

Abundant 

Abundant 

Good 

Good 

Poor 



In order to test the infecting power of the different cultures, three 
flowerpots of alfalfa plants grown in sterile soil were inoculated with 
the organism from each test tube. Four weeks after inoculation the 
plants were examined for nodule development. The results of the in- 
oculations are presented in table 8. Fifty-eight plants in flowerpots 
were used as controls. These plants were grown in sterile soil and were 
not inoculated. The control plants were distributed among the other 



44 



Martin J. Prucha 



plants in order to see to what extent an infection from neighboring flower- 
pots may take place. 



TABLE 8. 



Infecting Power of Various Cultures. B. radicicola Was Propagated 
for Three Weeks on the Different Media 



Medium 



Number Total 

of number 
plants of 

inoculated nodules 



Number 

of 
nodules 
per plant 



Medium 334, 10 cc. 



+ 

+ 0.1 cc. N/1 HC1 

+ 0.5 cc. N/1 HC1 

+ 1.0 cc. N/1 HC1 

+ 1.5 cc. N/1 HC1 

+ 2.0 cc. N/1 HC1 

+ 3.0 cc. N/1 HC1 

+ 

+ 0.1 cc. N/1 NaOH.. .. 

+ 0.5 cc. N/1 NaOH.. . . 

+ 1 Occ. N/1 NaOH... 

+ 1.5 cc. N/1 NaOH.. .. 

+ 2.0 cc. N/1 NaOH.. .. 

+ 3.0 cc. N/1 NaOH.. . . 

+ 0.1 per cent cane sugar 

+ 0.5 per cent cane sugar 

+ 10 per cent cane sugar 

+ 1.5 per cent cane sugar 

+ 2.0 per cent cane sugar 

+ 3.0 per cent cane sugar 

+ 4.0 per cent cane sugar 

+ 6.0 per cent cane sugar 

-f- 8.0 per cent cane sugar 
+ 10.0 per cent cane sugar 

Medium 335, 10 cc. 

+ 

+ 0.1 cc. N/1 HC1 

+ 0.5cc. N/1 HC1 

+ 1.0 cc. N/1 HC1 

+ 1.5cc. N/1 HC1 

+ 2 Occ. N/1 HC1 

+ 3 Occ. N/1 HC1 

+ 0.1 cc. N/1 NaOH 

+ 0.5cc. N/1 NaOH 

+ 1.0 cc. N/1 NaOH 

+ 1.5 cc. N/1 NaOH 

+ 2.0cc. N/1 NaOH 

+ 3.0cc. N/1 NaOH 



42 
50 
43 
32 
38 
53 
35 
37 
43 
48 
47 
34 
27 
27 
46 
35 
32 
48 
38 
29 
35 
33 
33 
30 



265 

320 

188 



6 

22 
7 
184 
275 
234 
304 
168 

94 

75 
182 
124 
117 
198 
140 
125 
138 
132 
110 

85 



3 

4 

3 

2 
4 
2 

4 
9 
6.5 
4.9 
3.5 
2.8 
4.0 
3.5 



29 
34 
50 
68 
50 
56 
58 
33 
32 
30 
36 
30 
21 



132 

183 

6 



22 

5 

4 

186 

131 

212 

284 

180 

109 



4.6 

5.4 

0.1 



0.4 

0.1 

0.1 

5.6 

4.1 

7.1 

7.9 

6.0 

5.2 



Studies of Bacillus Radio cola of Canada Field Pea 45 

TABLE 8 (concluded) 



Medium 



Number 

of 

plants 

inoculated 



Total 
number 

of 
nodules 



Number 

of 
nodules 
per plant 



Medium 337, 10 cc. 

+ 

+ 0.1 cc. N/1 HC1.. 
+ 0.5cc. N/1HC1... 
+ 1.0 cc. N/1 HC1.. 
+ 1.5 cc. N/1 HC1... 
+ 2.0cc. N/1 HC1... 
+ 3.0cc. N/1 HC1.. 
+ 0.1 cc. N/1 NaOH 
+ 0.5cc. N/1 NaOH 
+ 1.0 cc. N/1 NaOH 
+ 1.5 cc. N/1 NaOH 
+ 2.0cc. N/1 NaOH 
+ 3.0cc. N/1 NaOH 

Controls 



23 
32 
37 
36 
36 
33 
32 
26 
27 
39 
31 
32 
33 



54 

50 

110 

77 

23 

94 

132 

80 

120 

174 

114 

154 

87 



2.3 
1.6 
3.0 
2.1 
0.6 
2.8 
4.1 
3.1 
4.4 
4.5 
3.7 
4.8 
2.6 



735 



"624 



0.8 



* Nodules were present in only thirteen out of fifty-eight pots 



Results 
The extent of multiplication and the description of the morphological 
characters of the organisms in the various media are summarized in table 7. 
No visible increase in the number of organisms was found when 1 cubic 
centimeter or more of the normal solution of HO was added to 10 cubic 
centimeters of each medium. The few organisms found on these slopes 
were probably those that were introduced when the slopes were inoculated. 
In medium 337 slight multiplication took place on slopes of 10 cubic 
centimeters of the medium plus 1 cubic centimeter of the normal solution 
of HC1. The best multiplication occurred in media 335 and 337 when 
0.1 to 1 cubic centimeter of the normal solution of NaOH was added 
to 10 cubic centimeters of the medium. This was particularly noticeable 
in medium 337. The addition of 3 cubic centimeters of the normal solu- 
tion of NaOH to 10 cubic centimeters of the medium practically inhibited 
multiplication. The bacteroids developed more readily when NaOH was 
added. 



46 Martin J. Prucha 

The addition of sugar to medium 334, up to 10 per cent, caused more 
rapid multiplication and also the development of bacteroids. 

In medium 334 multiplication of the organisms was very slow and only 
a few bacteroids developed in the three weeks. In medium 337 multipli- 
cation was abundant and bacteroids developed. 

The results of inoculation are given in table 8. There were two hundred 
and eight flowerpots, which were crowded together because of lack of 
space. Fifty-eight flowerpots contained control plants, which were 
scattered among the inoculated pots. The plants in forty-five of the 
control pots developed no nodules; thirteen of the control pots were 
contaminated, and these were located mostly among the flowerpots 
inoculated with the organisms grown on medium 337. This contamination 
occurred in spite of the precautions taken to prevent the organisms from 
being carried from one flowerpot to another when the plants were watered. 
In these experiments and in others not reported in this paper, it was found 
that when a large number of flowerpots were used at one time it was 
difficult to prevent infection from other sources than that of the inoculating 
material. This was particularly true in the case of those legumes that 
produce an abundance of nodules in the soil of this region, and when 
the plants were allowed to grow for longer periods than three weeks. 
The results in this experiment are marred by a certain amount of con- 
tamination. The data in table 8 are so arranged as to show the total 
number of nodules on all the plants inoculated with the same material, 
and also to show the average number of nodules to each of these plants. 
Basing the conclusions on the mere number of nodules overemphasizes 
the importance of the contamination. When the plants were examined, 
and the size, location, and evenness of distribution of the nodules among 
the plants in the same flowerpot were noted, in addition to their total 
number, much more reliable evidence was obtained. In view of this, 
the following conclusions seemed to be warranted: 

When B. radicicola of alfalfa is propagated on media 334, 335, and 
337 and kept for three weeks, multiplication of the organism takes place 
and the infecting power is not lost. A slight reduction in infecting power 
seems to be apparent on medium 337. The addition of 0.1 cubic centimeter 
of the normal solution of HC1 to 10 cubic centimeters of each of these 
media very slightly diminished the amount of growth, but the infecting 
power was not affected. When 0.5 cubic centimeter of the acid solution 



Studies of Bacillus Radicicola of Canada Field Pea 47 

was added, the amount of growth was decidedly reduced on media 334 
and 335 and the infecting power also was reduced. The influence of this 
amount of acid in medium 337 was not so pronounced. The addition 
of 1 cubic centimeter or more of the acid solution completely inhibited 
the growth and the infecting power of the organism on all three 
media. 

The addition of 0.1, 0.5, 1, 1.5, and 2 cubic centimeters, respectively, of 
the normal solution of NaOH, to 10 cubic centimeters of each of the media, 
increased the amount of growth and also the effectiveness of the inoculation. 
The addition of 3 cubic centimeters of the normal solution of NaOH 
reduced the amount of growth and the infecting power. The heaviest 
growth took place on medium 337 to which the above amounts of NaOH 
were added, but the plants inoculated with these cultures did not produce 
as many nodules as did the plants inoculated with the organisms propa- 
gated on media 334 and 335 — a fact that suggests the possibility of 
a reduction in infecting power. 

The addition of cane sugar to medium 334 in the amounts indicated 
in table 8 has a beneficial influence on the multiplication of the organism. 
The infecting power does not seem to be affected by it. All the cultures 
in which any multiplication was observed produced positive inoculation, 
so that the infecting power of the organism was not destroyed. The 
variations in the number of nodules on the plants inoculated by the 
different cultures seem to indicate that the organisms propagated on 
medium 337 partly lost their infecting power. This measure of infecting 
power, however, is not accurate, and other explanations might easily 
be supplied. The most noticeable point was that positive inoculation 
was produced by all the cultures in which any multiplication took place, 
so that the growth and the infecting power seem to run parallel. 

experiment 12 
influence of some other media 
The organism, isolated as described in Part I, was kept in the laboratory 
for two years on agar slopes, medium 335, before this experiment was 
started. During this time the organism was continually exposed to 
diffused light and to the ordinary variations in temperature and humidity. 
The organism was then propagated on agar slopes, medium 334, to which 
various substances were added as is indicated in tables 9 and 10. Twelve 



48 



Martin J. Prucha 



TABLE 9. Description of Three-Weeks-Old Growth of B. radicicola on Agar 

Slopes 



Medium 334 with 



Description of growth 



2 per cent cane sugar 

2.0 per cent dextrose 

2 . per cent levulose 

2 per cent lactose 

2 . per cent galactose 

10.0 per cent cane sugar 

20 . per cent cane sugar 

40 . per cent cane sugar 

2 . per cent glycerin 

2.0 per cent mannite 

0. 1 per cent asparagin 

1 . per cent salicin 

0.5 per cent amygdalin 

. 5 per cent resorcin 

0.2 per cent phloroglucin 

0.5 per cent potassium oxalate 

0.5 per cent potassium citrate. 

0. 1 per cent potassium nitrate 

0.2 per cent potassium nitrate 

0.6 per cent potassium nitrate 

0.1 per cent calcium nitrate. . 

0.2 per cent calcium nitrate. . 

0.6 per cent ca cium nitrate. . 

1 . per cent gelatin 

5 . per cent gelatin 

1 .0 per cent Witte's peptone. . 

2 per cent Witte's peptone.. 
5 per cent Witte's peptone. . 
0.2 per cent Merck's peptone. 
1 .0 per cent Merck's peptone. 

3 per cent Merck's peptone. 



Good, watery 

Good, watery 

Not visible 

Not so good as with cane sugar 

Good, watery 

Good, watery 

Good, watery 

Slight, very transparent 

Good, watery 

Good, watery 

Fair 

Fair, whitish, not watery 

Fair, whitish 

Hardly visible 

Not visible 

Not visible 

Not visible 

Poor, fluorescent, opaque 

Poor, fluorescent, opaque 

Poor, fluorescent, opaque 

Medium, very opaque, fluorescent, tough, brownish 

Medium, very opaque, fluorescent, tough, brownish 

Medium, very opaque, fluorescent, tough, brownish 

Medium, juicy, opaque 

Poor 

Abundant, juicy 

Abundant, juicy 

Not visible 

Abundant, fluorescent, whitish to brownish 

Abundant, fluorescent, whitish to brownish 

Not visible 



tubes of each medium were prepared, and after being sterilized they 
were sloped and inoculated. 

. The following substances were used alone as media: soy bean hay, 
ground; soy bean roots, ground; Canada field pea hay, ground; Canada 
field pea roots, ground; Canada field pea seeds, ground; compost (well- 
decomposed cow feces), ground; partly decomposed cow feces, ground; fresh 
cow feces, ground; corn meal; sawdust; wheat bran; wheat middlings; 
sandy soil; muck; cornstarch. These substances were dried at 100° C, 



Studies of Bacillus Radicicola of Canada Field Pea 



49 



ground fine, and then introduced into test tubes 25 x 180 millimeters in size. 
Each tube was filled to one-third its capacity. Twelve test tubes were 
prepared for each medium, plugged with cotton, and sterilized in the 
autoclave for one hour at 15° C. For inoculation 10 cubic centimeters 
of sterile water in which the organisms were suspended was added to each 
test tube. At the end of two weeks the test tubes were sealed with 
paraffin in order to reduce evaporation. All the cultures were kept in 
the laboratory at room temperature. 

Three tests were made in order to determine the infecting power of the 
organism propagated on the various media. The first test was made at 
the end of the third week, the second at the end of the tenth week, and 
the third at the end of the twentieth week. In these tests one test tube 
was taken irom each of the media, the total number of organisms in each 
of these tubes was determined by the plate method, and Canada field pea 
plants were inoculated. 

The plants were grown in sterilized sandy soil in flowerpots, three 
flowerpots being inoculated with each culture. When the plants were 
three weeks old they were examined and the nodules on each plant were 
counted. In table 9 is given a description of the three-weeks-old growth 



TABLE 10. 



Number of Organisms in the Various Media at the Time the Tests 
Were Made 



Medium 



Number of organisms per gram 



Cultures 
three weeks old 



Cultures 
ten weeks old 



Cultures 
twenty- 
weeks old 



Soy bean hay 

Soy bean roots 

Canada field pea hay 

Canada field pea roots 

Canada field pea seeds 

Compost 

Partly decomposed cow feces . 

Fresh cow feces 

Corn meal 

Sawdust 

Wheat bran 

Wheat middlings 

Sandy soil 

Muck 

Cornstarch 



1,500, 

10,000, 

340, 

120, 

GO, 

7,200, 

32, 

10,000, 

4,000, 

60, 

620, 

200, 






Few 
000,000 
000,000 
000,000 
000,000 
000,000 
000,000 
000,000 
000 , 000 
000,000 
000,000 
000,000 
000,000 









306,000,000 

185,000,000 

75,000,000 

75,000,000 

440,000,000 

Contamination 

3,000,000 

370,000,000 

390,000,000 

37,000,000 

222,000,000 

55,000,000 



120,000,000 

3,240,000,000 

810,000,000 

97,000,000 

3,500,000 

600,000,000 

420,000,000 

20,000,000 
240,000,000 

97,000,000 



50 



Martin J. Prucha 



TABLE 10 (concluded) 



Number of organisms on one agar slope 



Medium 334 with 



Cultures 
three weeks old 



Cultures 
ten weeks old 



Cultures 

twenty 

weeks old 



2 Oper 
10.0 per 
20.0 per 
40.0 per 
2 Oper 
2.0 per 
2.0 per 
2.0 per 
2.0 per 
2.0 per 
. 1 per 
1 . per 
. 5 per 
. 5 per 
. 2 per 
. 1 per 
0.2 per 
. 6 per 
. 1 per 
. 2 per 
. 6 per 
1.0 per 
5 per 
1 per 
2.0 per 
5 per 
0.2 per 
1 . per 
3 . per 
. 5 per 
. 5 per 



cent cane sugar 

cent cane sugar 

cent cane sugar 

cent cane sugar 

cent dextrose 

cent levulose 

cent lactose 

cent galactose 

cent glycerin 

cent mannite 

cent asparagin 

cent -salicin 

cent amygdalin 

cent resorcin 

cent phloroglucin 

cent potassium nitrate, 
cent potassium nitrate, 
cent potassium nitrate, 
cent calcium nitrate. . . 
cent calcium nitrate . . . 
cent calcium nitrate. . . 

cent gelatin 

cent gelatin 

cent Witte's peptone . . 
cent Witte's peptone . . 
cent Witte's peptone. . 
cent Merck's peptone. . 
cent Merck's peptone. . 
cent Merck's peptone. . 
cent potassium oxalate, 
cent potassium citrate . 



No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 
No count 



made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 
made 



300,000,000 

3,000,000 

1,000,000 

16,000,000 

31,000,000 

400,000 

Plates spoiled 

3,000,000 

250,000,000 

300^000^000 

No colonies 

250,000,000 

No colonies 

Few 

120,000,000 

74,000,000 

259,000,000 

280,000,000 

150,000,000 

400,000,000 

150,000,000 

50,000,000 

50,000 

Contaminated 

No colonies 

22,000,000 

300,000,000 

No colonies 

No colonies 

No colonies 



90,000,000 

5,000,000 

10,000 

No colonies 

52,000,000 

No colonies 

180,000 

600,000 

20,000,000 

25,000,000 

108,000,000 

No colonies 

No colonies 

No colonies 

No colonies 

72,000,000 

224,000,000 

144,000,000 

486,000,000 

108,000,000 

130,000,000 

64,000,000 

Plates spoiled 

216,000,000 

648,000,000 

No colonies 

8,000,000 

324,000,000 

No colonies 

No colonies 

No colonies 



of B. radicicola on the agar slopes to which the various chemicals were 
added. The number of organisms in the cultures on the various media 
at the time they were tested for their efficiency to produce nodules is given 
in table 10. The results of the inoculations are shown in tables 11, 12, 
and 13. 

Results 

Growth and number of organisms (tables 9 and 10) 

Bacillus radicicola of Canada field pea produces scant growth on agar 



Studies of Bacillus Radicicola of Canada Field Pea 51 

slope medium 334. Some of the substances that were added to this 
medium retarded or completely inhibited the growth; others had no 
appreciable effect on the growth; and still others caused a decided increase 
in the growth as compared with that on medium 334 alone. (Table 9.) 
No visible growth was produced when the following substances were 
added: levulose 2 per cent, phloroglucin 0.2 per cent, potassium oxalate 
0.5 per cent, potassium citrate 0.5 per cent, Witte's peptone 5 per cent, 
Merck's peptone 3 per cent. The growth in the remaining cultures 
was watery, almost transparent, very viscous, especially in the presence 
of sugars. 

The number of organisms on the agar slopes was determined when the 
cultures were ten and twenty weeks old, and in the case of the substances 
in which the organism was propagated the determination was made at 
three, ten, and twenty weeks. The data in table 10 show a wide variation; 
but in general, wherever a visible amount of growth appeared on the agar 
slopes large numbers of organisms were found. At the end of ten weeks 
no organisms were found in the presence of salicin 1 per cent, resorcin 
0.5 per cent, Witte's peptone 5 per cent, Merck's peptone 3 per cent, 
potassium oxalate 0.5 per cent, and potassium citrate 0.5 per cent. At 
the end of twenty weeks, in addition to the above no organisms were 
found in the presence of care sugar 40 per cent, levulose 2 per cent, amyg- 
dalin 0.5 per cent, and phloroglucin 0.2 per cent. 

Very large numbers of the organism were found on most of the sub- 
stances that were ground and used as media. Wheat bran and ground 
Canada field pea seeds each had 10,000,000,000 organisms per gram at 
the end of three weeks. The organisms remained in a vigorous condition 
on these media for twenty weeks, as judged by the development of colonies 
and by the results of inoculation of the plants. In soy bean hay and 
soy bean roots no multiplication took place, and the organisms introduced 
at the time of seeding these two media were found to be dead at the end 
of three weeks. 

First test of injecting power {table 11) 

Three flowerpots were inoculated with each culture, these cultures 
being three weeks old when used. The inoculated plants were kept in 
the greenhouse and were examined for nodule development three weeks 
after inoculation. The data are presented in table 11 : 



52 



Martin J. Prucha 



TABLE 11. Infecting Power of Cultures Three Weeks Old 



Medium 334 with 



2 . per cent cane sugar 

2 . per cent dextrose 

2 . per cent levulose 

2 . per cent lactose 

2 . per cent galactose 

10 . per cent cane sugar 

20 per cent cane sugar 

30 . per cent cane sugar 

10 ( * per cent cane sugar 

1 . per cent salicin 

. 5 per cent amygdalin 

. 5 per cent resorcin 

. 2 per cent phloroglucin 

0.5 per cent potassium oxalate 

0.5 per cent potassium citrate 

. 1 per cent potassium nitrate 

. 2 per cent potassium nitrate 

. 6 per cent potassium nitrate 

. 1 per cent calcium nitrate 

. 2 per cent calcium nitrate 

. 6 per cent calcium nitrate 

1 . per cent Witte's peptone 

2.0 per cent Witte's peptone 

5.0 per cent Witte's peptone 

0.2 per cent Merck's peptone 

1 . per cent Merck's peptone 

3 . per cent Merck's peptone 

Other media 

Soy bean hay 

Soy bean roots 

Canada field pea hay 

Canada field pea roots 

Sandy soil 

Muck 

Sawdust 

Wheat bran 

Wheat middlings 

Corn meal 

Ground field peas 

Compost 

Partly decomposed cow feces 

Fresh cow feces 

Controls 

Controls 

Controls 

Controls 

Controls 



Nodules 

in 
flower- 
pot 1 

Many 

Many 

None 

Many 

Many 

Many 

Few 

Few 

Few 

Many 

Many 

None 

Few 

None 

None 

Many 

Many 

Few 

Many 

Many 

Many 

Many 

Few 

None 

Many 

Many 

None 



Nodules 

in 
flower- 
pot 2 



None 
Few 
Few 
Many 
Many 
Many 
Many 
Many 
Many 
Many 
Many 
Many 
Many 
Many 
None 
None 
None 
None 
None 



Many 
Many 
None 
Many 
Many 
Many 

Few 

Few 

Few 
Many 

Few 
None 

Few 
None 
None 
Many 
Many 

Few 
Many 
Many 
Many 
Many 

Few 
None 
Many 
Many 
None 



Nodules 

in 
flower- 
pot 3 



None 


None 


Few 


Few 


Few 


Few 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


Many 


None 


None 


None 


None 


None 


None 


None 


None 


None 


None 



Studies of Bacillus Radicicola of Canada Field Pea 



53 



As far as could be ascertained by these results, there was no reduction 
in the infecting power of the organisms when they were propagated and 
kept on the above media for three weeks. When these results are com- 
pared with the data in tables 9 and 10, it is seen that those cultures in 
which no visible growth took place produced either no inoculation or very 
poor inoculation, and that the cultures in which the organisms multiplied 
produced good inoculation. 

Second test of infecting power (table 12) 

In this test the cultures were ten weeks old. The exact number of 
nodules on each plant was counted, and from these figures calculations 
were made of (1) the total number of nodules in each flowerpot, (2) the 
average number of nodules per plant in each flowerpot, and (3) the average 
number of nodules per plant of all the plants inoculated with the same 
culture. It was hoped that the averages would give a more nearly ac- 
curate measure of the infecting power of the different cultures. The data 
are given in table 12. Fifteen plants were inoculated with each culture, 
five plants being grown in each flowerpot. 



TABLE 12. Infecting Power of Cultures Ten Weeks Old 



"3 

u 

a 

p 
p 

>> 


« s 

05 If 

O O 

a ft 

■- & 


"3 

V 

X> 

S 

P 

a 

bo 
° & 

"o3 g 

►J 


Num ber of 
nodules on 
each plant 


3 

T3 

a o 

v, ft 

S3 g 

3 -^ 
c o3 
— V 

■*> (3 


Average number of nod- 
ules per plant in each 
flowerpot 


number of nod- 
er plant pro- 
sy the culture 


o V 

03 P 

t- -*> 


r— 1 

a 

03 

5 


a 

03 

5 


co 

13 
o3 

S 




a 

03 

5 


Average 
ules p 
duced 


1 


Medium 334 -\- 2 per cent 
cane sugar 


1 

2 
3 


14 
22 


4 
10 


1 

2 



9 


12 
12 


31 
55 


6.2 
11.0 


8.6 




















2 


Medium 334 + 2 per cent 
cane sugar 


4 
5 
6 


6 
12 
14 


.0 

4 
7 


3 

15 
6 


10 

12 

2 


4 
10 

7 


23 
53 
36 


4.6 
10.6 

7.2 


7.5 


4 


Medium 334 + 10 per cent 
cane sugar 


7 
8 
9 


5 
10 
10 


7 

8 

15 


14 

14 
18 


12 
8 
5 


12 

"s 


50 
40 
53 


10.0 
10.0 
10.6 


10.2 



54 



Martin J. Prucha 



TABLE 12 (continued) 



o 

u 

s 

a 

a 


05 of 

|2 

P CO 

■S & 

cy u 


"o 

u. 
CD 

a 

a 

CO 

2 o 
-§« 


Number of 
nodules on 
each plant 


CO 

2*> 

a o 

°£ 

a 

C o3 
— v 

$ a 
o -rH 
H 


Average number of nod- 
ules per plant in each 
flowerpot 


number of nod- 
er plant pro- 
ay the culture 


° "a 


5 


CO 

C 

03 

5 


CO 

p 

o3 


a 
5 


c 

03 
5 


CD -rt 
be o> 

< 


6 


Medium 334 -f- 20 per cent 
cane sugar 


10 

11 

12 


8 

15 

4 


1 

5 




8 
1 
4 


2 3 
5 



22 

26 

8 


4.4 
5.2 
1.6 


3.7 


7 


Medium 334 + 40 per cent 
cane sugar 


13 
14 

15 


11 
18 

8 


8 

12 

3 


9 

8 
8 


5 .... 
16 ... . 

8 6 


33 
54 
33 


8.2 

13.5 

6.6 


9.2 


8 


Medium 334 + 2 per cent 
dextrose 


16 
17 

18 



10 

4 


14 
12 

8 


15 

14 

4 


10' 9 

7 .... 

i- 


48 
43 
22 


9.6 

10.7 

5.5 


8.7 


9 


Medium 334 -f- 2 per cent 
lactose 


19 

20 
21 



3 




3 
1 

6 


1 


5 


5 .... 
2 3 
0j 


9 

9 
11 


2.2 
1.8 

2.2 


2.1 


10 


Medium 334 + 2 per cent 
galactose 


22 
23 
24 


5 

10 
8 


8 

11 

9 


7 
16 
10 






20 
41 
56 


6.7 
10.2 
11.2 






4 
15 


14 


9.7 


12 


Medium 334 -f- 2 per cent 
glycerin 


28 
29 
30 


3 
15 


2 
15 


16 
20 


14 14 
10 ... . 


49 
60 


9.8 
15.0 


12.1 


















13 


Medium 334 -f- 0.5 per cent 
potassium oxalate 


31 
32 
33 








2 




3 



o 1 

4 . ... 




9 





2.2 



0.6 


14 


Medium 334 + 0.2 per cent 
phloroglucin 


34 
35 
36 





1 




3 





2 


3 .... 
.... 
5 15 


3 



26 


0.7 



5.2 


2.2 


15 


Medium 334 + 0.5 per cent 
resorcin 


37 
38 
39 


5 
1 



5 
4 



5 




8 .... 
6.... 



23 

11 




5.7 

2.7 



2.6 


16 


Medium 334 + 1 per cent 
salicin 


40 
41 
42 




10 




2 


2 


4 





5 

4 


3 
12 


9 

27 
6 


1.8 

5.4 
1.5 


3.0 


17 


Medium 334 -4- 0.5 per cent 
amygdalin 


43 
44 
45 



10 
14 


8 
15 
20 


14 
24 
10 


2 
15 


16 
8 


40 
72 
44 


8.0 
14.4 
14.7 


12.0 



Studies of Bacillus Radicicola of Canada Field Pea 



55 





TABLE 


12 (c 


ontin 


ied) 












u 

V 
X! 

£ 

3 

c 


~5 -a 

G3 If 

^ & 
o o 

2 ft 
& » 

d o3 

.2 £ 
£ « 
.2 "3 
-3-S 


Laboratory number of 
flowerpots 


Number of 
nodules on 
each plant 


05 
JO 

3 

Co 

° as 

•21 


C 03 
_ <0 

S 3 
O"- 

H 


Average number of nod- 
ules per plant in each 
flowerpot 


lumber of nod- 
er plant pro- 
>y the culture 


O V 

rt 3 

° "3 


-^ 

a 

o3 


a 


CO 

a 

03 


3 
o3 

5 


a 

03 

5 


Average 
ules p 
duced 


18 


Medium 334 -f 0.1 per cent 
asparagin 


46 
47 

48 


16 10 
10 5 

6 


6 

8 

12 


18 

10 

2 


10 

"10 


60 
33 
30 


12.0 
8.2 
6.0 


8.8 


19 


Medium 334 + 0.1 per cent 
potassium nitrate 


49 
50 
51 


7 3' 10 

12 5; 6 

13 15 


12 




15 

5 


47 
23 
33 


9.4 

4.6 
6.6 


6.9 


20 


Medium 334 + 0.2 per cent 
potassium nitrate 


52 
53 
54 


2 
1 



2 6 

8 8 
2| 1 


9 
12 

8 


10 

1 

10 


29 
30 
21 


5.8 
6.0 
4.2 


5.3 


21 


Medium 334 + 0.G per cent 
potassium nitrate 


55 
56 
57 


10 
13 

12 


16 10 

16 2 

5 


18 
30 

2 


2 
12 


56 
61 
31 


11.2 

15.2 

6.2 


10.6 


22 


Medium 334 + 0.1 per cent 
calcium nitrate 


58 
59 
60 


3 

6 

8 


2 5 
6| 3 
0, 14 


10 

10 

5 


8 

8 

12 


28 
33 
39 


5.6 

6.6 

7.8 


6.7 


23 


Medium 334 + 0.2 per cent 
calcium nitrate 


61 
62 
63 


12 

6 

16 


12 

6 

12 


10 
20 
20 


5 
18 

8 


14 
22 
16 


53 
72 
72 


10.6 
14.4 
14.4 


13.1 


24 


Medium 334 + 0.G per cent 
calcium nitrate 


64 
65 
66 


12 
3 



4 
2 



3 

7 
6 




7 
10 


10 
9 
5 


29 
28 
21 


5.8 
5.6 
4.2 


5.2 


25 


Medium 334 + 0.5 per cent 
potassium citrate 


67 
68 
69 


4 




2 

8 


3 





1 




"o 




10 




2.5 




0.7 


26 


Medium 334 + 0.2 per cent 
Merck's peptone 


70 

71 
72 


4 





7 8 
2 5 
5 


4 
5 

7 


4 

"10 


27 
12 
22 


5.4 

3.0 
4.4 


4.4 


27 


Medium 334 + 1 per cent 
Merck's peptone 


73 

74 
75 



20 

7 


2 8 

7 

8 


7 



12 


14 

8 
2 


31 
35 

29 


6.2 

7.0 

5.8 


6.3 


28 


Medium 334 + 1 per cent 
Witte's peptone 


76 

77 
78 


14 

14 

5 



35 
30 


10 
20 
25 



30 

7 


15 


39 
99 
67 


7.8 
24.7 
16.7 


15.8 



56 



Martin J. Prucha 





TABLE 


.2 {continued) 












"3 

s 

3 

c 


"S "3 
3 43 

03 If 

1 2 

£ to 

fl 03 

S"o 

•3-S 
43 U 


y 

43 

a 

3 

a 

bo 

° & 

"5 »> 

■a* 


Number of 
nodules on 
each plant 


Total number of nodules 
in each flowerpot 


Average number of nod- 
ules per plant in each 
flowerpot 


number of nod- 
er plant pro- 
sy the culture 


O 03 

** 3. 

° "3 


a 
5 


3 
03 

5 


a 

03 
5 


a 

03 


c 

03 
5 


Average 
ules p 
duced 


29 


Medium 334 + 2 per cent 
Witte'a peptone 


79 
80 
81 




2 



2 
3 

1 



4 
5 


4 






1 


6 

10 

6 


1.2 
2.0 
1.5 


1.6 


30 


Medium 334 + 5 per cent 
Witte's peptone 


82 
83 
84 



2 
















4 










6 





1.2 




0.4 


31 


Medium 334 + 5 per cent 
gelatin 


85 
86 
87 


10 
2 



14 

8 

18 


16 

10 

5 


35 
16 
10 


34 

6 

25 


109 
42 

58 


21.8 

8.4 

11.6 


13.9 


32 


Soy bean roots, ground 


88 
89 
90 





2 









1 



3 

7 





"o 


3 

8 
2 


0.6 

2.0 
0.4 


0.9 


33 


Soy bean hay, ground 


91 
92 
93 









































34 


Canada field pea roots, 
ground 


94 
95 
96 




8 
3 



10 
20 


5 

6 
5 


8 
12 

8 


6 
10 

4 


19 
46 
40 


3.8 
9.2 
8.0 


7.0 


35 


Canada field pea hay, ground 


97 
98 
99 


12 




5 




2 

7 



8 
2 



"l 



27 

10 




6.7 
2.0 



2.6 


36 


Sawdust 


100 
101 
102 


1 

10 










7 




6 








1 

23 




0.2 
4.6 



1.6 


37 


Corn meal 


103 
104 
105 










1 



















1 




0.2 


0.1 


38 


Cornstarch 


106 
107 
108 


2 
18 

7 


6 

6 

15 



2 

8 


12 
12 
15 


16 
"20 


36 
38 
65 


7.2 

9.5 

13.0 


9.9 


39 


Wheat bran 


109 
110 
111 


18 
8 




8 


8 
8 


8 
14 


6 
8 


40 
46 


8.0 

9.2 


8 6 



Studies of Bacillus Radicicola of Canada Field Pea 57 





TABLE 


12 (cc 


ncluo 


ed) 












o 

s> 

.a 
S 

3 

a 
>> 


s^ 

05 If' 

■1 2 

-3 a, 

£ . 

3 =3 
•= & 

S.S 

.go 

-a-s 


£ 

3 
3 

05 

O £• 

2 o 


o g-g 

^3 h 

5 2 * 

3 3 <U 


CO 

CD 

O +» 

a 2 

o 5 

n is 

§"3 

_ a> 

J 3 

H 


Average number of nod- 
ules per plant in each 
flowerpot 


Average number of nod- 
ules per plant pro- 
duced by the culture 


o « 

"S 3 

o "3 

-1 




3 
S3 


CO 

3 


3 
o3 

5 


"S 

03 


40 


Wheat middlings 


112 
113 
114 


6 
20 
12 


18 

12 




20 
5 

7 


4 10 
6 10 
3 14 


58 
53 
36 


11.6 
10.6 

7.2 


9.8 


41 


Compost 


115 
116 
117 


16 
12 
10 


3 

4 

14 



15 
12 


19 4.7 

8 39 7.8 

16 8 60 12.0 


8.4 


42 


Partly decomposed cow feces 


118 
119 
120 


5 

6 

8 


1 

6 


7 

14 
3 


5 
8 
4 


10 33 6.6 

12 43 8.6 
14 35 7.0 


7.4 


43 


Fresh cow feces 


121 
122 
123 



3 

20 




7 
10 


7 
20 
12 




5 

12 


"l2 
10 


7 1.7 
47 9.4 
64 12.8 


8.4 


44 


Muck 


124 
125 
126 


10 
6 
3 


8 

6 

12 


10 12 

5 9 
8 3 


10 

5 


50 
26 
31 


10.0 
5.2 
6.2 


7.1 


45 


Sandy soil 


127 
128 
129 


22 
10 


14' 16 5 
8 4 6 


57 

28 


11.4 
5.6 


8.5 






1 








46 


Controls 


130 
131 
132 







o 1 

o; o 

3, 















3 




0.6 


0.2 


47 


Controls 


133 
134 

135 








o! o 



























.... 








The average numbers of nodules per plant for all the plants inoculated 
with each of the given cultures appear in the last column in table 12. In 
table 13 these numbers are rearranged in numerical order according to 
the average number of nodules per plant produced by each culture: 



58 



Martin J. Prucha 



TABLE 13. 



Average # Number of Nodules per Plant in Second Test, Arranged in 
Numerical Order 



Medium 



Organisms 

living or 

not living* 



Medium 334 + 1 per cent Witte's peptone. . . . 

Medium 334 + 5 per cent gelatin 

Medium 334 + 0.2 per cent calcium nitrate. . . 

Medium 334 + 2 per cent glycerin 

Medium 334 + 0.5 per cent amygdalin 

Medium 334 + 0.6 per cent potassium nitrate. 

Medium 334 + 10 per cent cane sugar 

Cornstarch 

Wheat middlings 

Medium 334 + 2 per cent galactose 

Medium 334 + 40 per cent cane sugar 

Medium 334 + 0.1 per cent asparagin 

Medium 334 + 2 per cent dextrose 

Medium 334 + 2 per cent cane sugar 

Wheat bran 

Sandy soil 

Compost 

Fresh cow feces 

Medium 334 + 2 per cent cane sugar 

Partly decomposed cow feces 

Muck 

Canada field pea roots 

Medium 334 + 0.1 per cent potassium nitrate. 
Medium 334 + 0.1 per cent calcium nitrate. . . 
Medium 334+1 per cent Merck's peptone. . . 
Medium 334 + 0.2 per cent potassium nitrate. 
M<' ium 331 + 0.6 per cent calcium nitrate. . 
Medium 334 + 0.2 per cent Merck's peptone . . 

Medium 334 + 20 per cent cane sugar 

Medium 331+1 per cent salicin 

Medium 334 + 0.5 per cent resorcin 

Canada field pea hay 

Medium 334 + 0.2 per cent phloroglucin 

Medium 334 + 2 per cent lactone 

Sawdust 

Medium 334 + 2 per cent Witte's peptone. . . . 

Soy bean roots 

Medium 334 + 0.5 per cent potassium citrate . 
Medium 334 + 0.5 per cent potassium oxalate. 
Medium 334 + 5 per cent Witte's peptone. . . . 

Controls (not inoculated) 

Corn meal (culture contaminated) 

Controls (not inoculated) 

Soy bean hay 



+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 





+ 
+ 













* + indicates living organisms; indicates no living organisms. 



Studies of Bacillus Radicicola of Canada Field Pea 59 

In making any deductions from the preceding data, it must be remem- 
bered that the plants in this and in the other experiments were grown 
under special conditions. If the number of nodules on the plants in this 
experiment were dependent only on the degree of infecting power of the 
organism, an excellent illustration of variation in the infecting power 
would here be shown. This variation would, in this case, be due to the 
nature of the medium in which the organism was propagated. 

That the number of nodules on a plant may be influenced by other 
factors than the infecting power of the organism has been shown in Part 
II of this paper, and also by experiments of other investigators. But 
the part that such other factors have played in this second test must be 
only a conjecture. 

In order that a nodule may be produced, it is necessary that at least 
one organism shall come in contact with the root, that it shall enter the 
tissue of the root, and that it shall multiply inside the tissue. At least 
six factors can be mentioned which may have been of some importance 
in this experiment in bringing about this result: 

1. The distribution of the organisms through the entire volume of the soil 
in each flowerpot. — The plant roots grow rapidly during the first three 
weeks after planting, and unless the organisms are evenly distributed 
through the soil a variation in the number of nodules might result if the 
plants are not allowed to grow for more than three weeks. Watering 
would tend to bring about an even distribution. All the flowerpots were 
watered twice each week, but the amount of water introduced into each 
pot was not measured — although it was uniformly constant — and be- 
cause of this the distribution of the organisms throughout the soil may 
not have been uniform in all the flowerpots during the first two weeks. 

2. The number of organisms introduced into the different flowerpots at the 
time of inoculation. — There is no available evidence to show how important 
this factor may be. That it may have exercised some influence seems 
highly probable. Reference to table 10 shows that there was a very 
great difference in the total number of organisms in the various cultures, 
and consequently the flowerpots inoculated with these cultures did not 
receive the same number of organisms. 

3. Multiplication of the organisms in the soil after inoculation. — Bacillus 
radicicola multiplies readily in the sterilized soil that was used for growing 
the plants. This does influence the total number and the distribution 



60 Martin J. Prucha 

cf the organisms in the soil. Moreover, if the infecting power of the 
organism were easily affected by the medium, it would be easily affected 
by the soil into which the organisms were introduced at the time of in- 
oculation. The result would be that in course of time the infecting power 
of the different organisms, although different at the beginning, would 
become the same for all the organisms. If this is true, the difficulty in 
measuring the infecting power of a given culture is very evident. 

4. Resistance of the -plant against the invasion of the organism. — Nothing 
is known of this in connection with the leguminous plants and the nodule- 
forming organism. If such a character does exist in the plants, it probably 
varies in the different individuals and would influence the number of 
nodules formed. 

5. Infection from other sources than the inoculating material. 

6. Infecting power of the organisms. 

It is evident from the above discussion that the number of nodules on 
plants may be the result of several factors operating simultaneously, and 
that it would be a difficult matter to determine their influence singly. 
The number of nodules, therefore, is not an accurate measure of the in- 
fecting power of the different cultures. Unfortunately, however, no other 
measure is available. In interpreting the above data, therefore, the 
limitations in the accuracy of the method must be borne in mind. 

Comparing the data in table 13 with those in table 10, a close relation 
may be observed between the condition of the cultures when ten weeks old 
and the number of nodules on the plants inoculated with these cultures. 
The following cultures had no living organisms: 

Medium 334 + 2 per cent Witte's peptone 

Medium 334 + 5 per cent Witte's peptone 

Medium 334 + 0.5 per cent resorcin 

Medium 334 + 1 per cent salicin 

Medium 334 + 0.5 per cent potassium oxalate 

Medium 334 + 0.5 per cent potassium citrate 

Medium 334 + 0.2 per cent phloroglucin (few organisms) 

Soy bean hay 

Soy bean roots 

Canada field pea hay 

Corn meal 

Reference to table 13 shows that the plants inoculated with these cultures 
produced three nodules or less per plant. Whether the few nodules found 



Studies of Bacillus Radicicola of Canada Field Pea 



61 



resulted from contamination or from a few surviving organisms not de- 
tected by the plating can only be conjectured. The remaining cultures 
all produced good inoculation, with the exception of the sawdust and 
the lactose culture, the average number of nodules per plant varying 
between 3.7 and 15.8. To what extent this variation in the number 
of nodules is due to the infecting power of the different cultures 
it is difficult to say with certainty; but that a part of the variation 
is due to other causes is evident from the data in the next to the last 
column in table 12. In this column the figures represent the average 
number of nodules per plant for every flowerpot. Since three flowerpots 
were inoculated with each culture, the extent of variation in the number of 
nodules in these pots must be due to other causes than the infecting 
power. A number of the more pronounced cases of variation in the 
number of nodules in the three respective flowerpots which were inoculated 
by the same culture are shown in table 14: 



TABLE 14. Variation in Average Number of Nodules in 


Some of the 


Flowerpots 


Inoculated with culture number 


Average number of nodules in 




Flowerpot 1 Flowerpot 2 


Flowerpot 3 


28 


24:7 
21.8 
15.2 
14.7 
13.5 


7.8 
8.4 
6.2 
8.0 
fi fi 


16.7 


31 


11 6 


21 


11 2 


17 


14 4 


7 


8 2 


38 


13.0 7.2 
12.8 1.7 
12:0 6.0 
12.0 f 4.7 
11.4 5.6 
11.0 fi 2 


9 5 


43 


9 4 


18 


8 2 


41... . 


7 8 


45 




1 




8 


10.7 

10.6 

10.0 

9.2 

5.2 


5.5 
4.6 
5.2 
3.8 
1.6 


9.6 


2 


7.2 


44 


6.2 


34 


8.0 


6 


4.4 







The three flowerpots inoculated with culture 43 had 12.8, 1.7, and 9.4 
nodules per plant, respectively. A similar relation exists between the 
other flowerpots considered in table 14. If the efficiency of these cultures 
were based on the number of nodules per plant, culture 43, for example, 



62 Martin J. Prucha 

would be either very efficient or much reduced in infecting power, depend- 
ing on which flowerpot was taken. 

It seems apparent, from the preceding discussion, that it is extremely 
difficult to measure any variations in the infecting power of different 
cultures. When the great danger of infection of plants grown in sterile 
soil but not under sterile conditions is considered, and also the number 
of other factors that may affect nodule development on plants, not many 
clear-cut conclusions can be drawn from the second test. If the infecting 
power is measured by the number of nodules, by their size, by the uni- 
formity of their distribution, and by their location, the following con- 
clusions seem reasonable: 

1. The cultures producing more than three nodules per plant are all 
efficient. 

2. If one culture produced 3.7 nodules per plant and another culture 
produced 15.8 nodules per plant, the belief that the latter culture possesses 
greater infecting power than the former is not justified. 

3. Some cultures produced no nodules, or only a few nodules confined 
to only one or two plants. Such cultures unquestionably lost their effi- 
ciency, but this loss of efficiency was parallel with the condition of the 
cultures. When no living organisms were found in a culture by using the 
plate method, such a culture produced no inoculation; and when living 
organisms were found, inoculation was produced. 

4. Propagating and keeping B. radicicola of Canada field pea for ten 
weeks on media rich in nitrogenous matter, such as wheat bran, wheat 
middlings, fresh cow feces, and potassium and calcium nitrates, did not 
destroy the infecting power of the organism. If any injury to infecting 
power was caused by these substances, it could not be detected by the 
methods used in this experiment. 

Third test of infecting power (table 15) 

Cultures twenty weeks old were employed in this experiment. In 
inoculating the plants a known number of organisms was introduced into 
each flowerpot. By correlating the relation between the number of or- 
ganisms used for the inoculation of each flowerpot and the number of 
nodules on the plants, it was hoped to find a more nearly exact measure 
of the infecting power of the organisms propagated in the different media. 
The results are summarized in table 15: 



Studies of Bacillus Radicicola of Canada Field Pea 



63 



TABLE 15. Infecting Power of Cultures Twenty Weeks Old 



sj 

5 
"3 

o 

"o 

u 

a> 

£ 
c 
>1 


"5 

s 

^- 

d a 
■- o 

E o. 

3 09 


PS 



<a 

*o 

u 
o 
X> 

s 

3 

a 
>> 

u 

o 
*J 

2 « 

u o 

•° ft 

oj ^ 


Number of nodules 
on each plant 


a 

"3 
-a 

d 



£ " 

X oj 

d« 

^M 

3 03 

o° 


u 

09 

ft 

09 



^0 

d * 

_ (6 



1° 

§ 09 

fl .2 

ti 
< 


U 09 

99 i- 

ftd 
09 d 
?J 

09 09 

■S3 

c ft 

SPd 

£ft 


6 

u 

at 

•" 0. 

09 49 

II 
d*" 

03 JS 

Mo 

O 09 
<_ O 
d 


OS 

o 

£■ 

s 

1-1 


□ 

03 


d 

OJ 

S 


CO 

d 
S 


■f 
d 

03 


d 

00 


CD 
-t-> 

d 

03 
S 


d 

09 

s 


|"8 


1 


Medium 334 + 2 per 
cent cane sugar. . . . 


1 
2 
3 


10J 6 
4 2 


5 


?, 


8 
4 






31 
10 
22 


6.2 
2.0 
5.5 


4.5 


324,000 
















324,000 




8 


14 






324,000 










3 


Medium 334 + 2 per 
cent cane sugar 


4 
5 
6 


10 
6 
8 


14 
1 
6 


6 






6 


: 




30 

7 

25 


7.5 

1.7 
5.0 


4.8 


259,200 









259,200 




R 






259,200 












4 


Medium 334 + 10 per 
cent cane sugar .... 


7 
8 
9 



































3.1 


90,000 




5 




5 


8.3 


90,000 
90,000 














5 


Medium 334 + 10 per 
cent cane sugar 


10 
11 
12 







5 





4 


6 








15 

6 


3.7 



1.0 


1.4 


5,000 





3 





n 






5,000 




3 







5,000 


6 


Medium 334 + 20 per 
cent cane sugar 


13 
14 
15 




6 




12 





8 




6 7 







13 

26 




2.6 

8.7 


3.0 


10 








10 








10 












7 


Medium 334 + 40 per 
cent cane sugar 


16 
17 
18 





































































8 


Medium 334 + 2 per 
cent dextrose 


19 
20 
21 


8 

4 


4 
6 


4 
6 




8 








16 
20 

4 


4.0 
5.0 
4.0 


4.4 


52,000 










52,000 










52,000 


















9 


Medium 334 + 2 per 
cent lactose 


22 
23 
24 


5 

S 

8 


4 
6 




12 


16 


6 

10 


8 
4 






"3 


35 
21 
34 


5.8 
3.0 

8.5 


5.3 


180 
180 
180 












10 


Medium 334 + 2 per 
cent galactose 


25 
26 
27 


12 


10 


6 
15 



16 








44 
43 
14 


11.0 
6.1 
2.3 


5.9 


60,000 




5 



4 



6 
3 



4 


5 

7 


8 


600 
600 


11 


Medium 334 + 2 per 
cent mannite 


28 
29 
30 


10 
12 

16 


1 
5 

8 




2 
12 


10 

8 


14 

2 


7 




42 
29 
36 


7.0 
5.8 
12.0 


7.6 


250,000 
2,500 








2,500 












12 


Medium 334 + 2 per 
cent glycerin 


31 
32 
33 


12 

8 
8 


15 

14 

9 


6 
15 

7 


7 
15 
11 


5 
16 
18 


10 
10 
14 


3 

8 


58 
86 
67 


8.3 
12.3 
11.2 


10.5 


2,000,000 
20,000 
20,000 


n 


Medium 334 + 0.5 per 
cent potassium oxa- 
late 


34 
35 
36 

















! 









5 




1.2 


0.3 








5 


o 





















14 


Medium 334 + 0.2 per 
cent phloroglucin 


37 
38 
39 













4 


0, 
8: 6 









18 




3.6 


1.6 



































< r 


Medium 334 + 0.5 per 
cent resorcin 


40 
41 
42 


























































Oj 

















it 


Medium 334 + 1 per 
cent salicin 


43 
44 
45 
























































64 



Martin J. Prucha 



TABLE 15 (continued) 



o 

a 

"3 

"o 

<u 

X 
5 


Medium in which B. radicicola 
was propagated 


o 

o 
en 

"S 

o 
X 
£ 

a 
>> 

u* 

o 

03 ni 
1° 
►J 








3 

a a 
a 

o" 3 - 

X 
Q> 03 

X • 

sg 

3 O 








o 

~3 

o 

a 

"o ^ 

o 

X fe 

3r2 

^x 

03 c3 


<v 

a 

ai 

J° 

a « 
^ s* 

o 
u m 

J2"S 

S3 cj 

3» 

55. S 

Ifa 

2 03 

> ^ 
< 


11 
o-2 
a 

«- >> 
o-a 

S e» 

■S3 
1-g 

a a 

la 

> ft 

< 


o 

"So 
— a 

£ * 

m O 

■~<X 

a 

M o 
fe o3 

«-, ° 


09 

c 

d 

- 


03 

5 


CN 

B 

E 


m 
a 

03 
5 


"3 

o3 

s 


a 

03 


CO 
-*^ 

a 
a 

S 


a 

03 

s 


11 

N 


17 


Medium 334 +0.5 per 
cent amygdalin 


46 
47 

48 

















o 



























































1M 


Medium 334 +0.1 per 
cent asparagin 


49 
50 
51 


5 
6 
8 


16 

15 



17 
12 
16 


18 
10 
12 


7 

6 

18 







63 
49 

58 


10.5 
9.8 
9.7 


10.0 


10,800,000 
108 , 000 




4 




108,000 


19 


Medium 334 + 0.1 per 
cent potassium 
nitrate 


52 
53 
54 


25 



20 


17 

8 

12 


6 
10 
10 




4 








48 
44 
50 


12.0 
8.8 
8.3 


9.5 


7,200,000 




26 

4 






72,000 









72,000 


20 


Medium 334 + 0.2 per 
cent potassium 
nitrate 


55 
56 
57 


3 

5 


5 
5 


3 



10 



5 


3 




29 
10 


4.8 
2.5 


3.9 


224,000 
224,000 








224,000 
















21 


Medium 334 + 0.6 per 
cent potassium 
nitrate 


58 
59 
60 




14 

6 


10 
3 
10 


12 
12 



4 


14 







40 
29 
18 


6.7 
9.7 
4.5 


6.7 


14,400,000 
144,000 




2 






144,000 










22 


Medium 334+ 0.1 per 
cent calcium nitrate 


61 
62 

63 


12 
2 

8 


12 
1 
4 


8 

7 

16 


•2 

9 



6 
5 
5 






40 
27 
47 


8.0 
4.5 
7.8 


6.7 


48,600,000 




3 

14 




486,000 
486,000 


23 


Medium 334 + 0.2 per 
cent calcium nitrate 


64 
65 
66 


2 
2 l 


1 
8 
3 


8 
5 
6 


14 

6 

14 


12 

16 

3 


16 

18 

8 


.... 1 53 
6 79 
l| 40 


8.8 

11.3 

5.7 


8.6 


10,800,000 
108,000 
108,000 


;>i 


Medium 334 + 0.6 per 
cent calcium nitrate 


67 
68 
69 


3 
13 


10 

7 
8 


8 

8 

10 


11 
9 
16 








35 
53 

59; 


8.7 

7.6 

11.8 


9.2 


13,000,000 




11 
12 


7 


8 


130,000 
130,000 




.... 






"f> 


Medium 334 + 0.5 per 


70 
71 
72 


12 

18 

1 


12 
8 



16 
4 

8 


4 
20 
12 


6 






50 
50 

28 


10.0 

12.5 

5.6 


9.1 


300,000 




cent potassium 
citrate 






3,000 




7 






3,000 










26 



B7 


Medium 334 + 0.2 per 
cent Merck's pep- 
tone 


73 
74 
75 


1 
3 

8 



5 



1 
12 




12 
12 
14 


10 







24 
32 

38 


4.0 
8.0 
6.3 


5.9 


800,000 
8,000 


6 


10 




8,000 


Medium 334 + 1 per 


76 

•77 
78 


2 
10 
12 


4 
12 
13 


10 
15 



14 
18 








30 
59 
25 


7.5 
11.8 
8.3 


9.5 


32,400,000 




cent Merck's pep- 
tone 


4 






324,000 








324,000 












28 


Medium 334 + 1 per 


79 
80 
81 


3 

4 
12 


7 
12 

8 


3 
10 

16 



12 


5 
14 


1 


18 
87 
36 


3.6 
12.4 
12.0 


9.4 


21,600,000 




cent Witte's peptone 


15 


20 


216,000 
216,000 
















29 Medium 334 4- 2 Der 


82 
83 
84 


12 

4 

10 


4 

14 


4 
12 
9 


10 

8 








30 
24 
33 


7.5 

6.0 

11.0 


7.9 


64,800.000 




centWitte's peptone 








648,000 




1 




648,003 












30 


Medium 334 + 5 per 
cent Witte's peptone 


85 
86 
87 


















2 














' "o 


2 




0.3 






0.1 







31 

1 


Medium 334 + 5 per 
cent gelatin 


88 
89 
90 


3 
5 
7 


2 
8 
8 


2 

7 

12 


10 
6 
14 


I 

5 


6 
2 




30 
36 
46 


5.0 
6.0 
9.2 


6.6 


6, 480, 000 
64,800 
64,800 



Studies of Bacillus Radicicola of Canada Field Pea 



65 



TABLE 15 (concluded) 



o 

1- 
£ 

"3 

'o 

-2 

£ 
3 
a 

>. 
c 
"5 

u 

o 

X 
r: 


Medium in which B. radicicola 
was propagated 


u 

0> 

is 

o 
en 

"o 
u 

ja 

c 

3 
fl 

>> 
o 

S3 m 

°o 

■§ ft 

Hi 








a 

"3 
■a 

fl fl 
J2 

'o a 
-fl 

i~ o 
01 e3 
„q oj 

s s 

3 O 








Total number of nodules in 
each flowerpot 


01 

ft 

GO 

o> 
1o 

"d Q, 

o fcr 

C 0) 
G O 

° -B 

■§! 

o H 

5 si 

< 


I - 0) 

"3 " 
~o & 
5 -9 
e~ 
^ >> 

fc"8 

■as 

3 O 
C ft 

< 




C o 

•"" ft 

u 
m o> 

c & 

oS o 

'fl* 

S3 .fl 
Mo 

o S 

•~ ° 
°fl 


fl 

cj 

Ph 


c 

03 
Ph 


CO 

fl 

Ph 


fl 

03 
Ph 


fl 

33 

PL, 


to 
e 

03 

Ph 


fl 

S3 

Ph 


11 


32 


Soy bean roots, ground 


91 
92 
93 















n 

























































33 


Soy bean hay, ground 


94 
95 
96 





































































34 


Canada field pea roots, 
ground 


97 
98 
99 







































































35 


Canada field pea hay, 
ground 


100 
101 
102 







































































30 




103 
104 
105 


15 
4 
5 














15 

26 

5 


15.0 
5.2 
5.0 


6.6 


350,000 




9 


5 


5 


3 






3,500 








3,500 




















37 




106 
107 
108 


6 

1 

12 


10 
2 
6 


8 
3 
8 


16 
12 

8 








40 
37 
35 


10.0 
6.2 

7.0 


7.5 


9 , 700 , 000 






5 

1 


14 . 


97,000 
97,000 


38 


Cornstarch 


109 
110 
111 


12 
5 
8 


7 
6 

7 


14 










33 
11 
29 


11.0 
5.5 
9.7 


9.1 


9,700,000 












97,000 




14 










97,000 














39 




112 
113 
114 


7 

1 

14 


12 

2 

22 


10 

22 












29 
33 
36 


9.7 

8.2 

12.0 


9.8 


60,000,000 






8 








600,000 










600,000 














40 


Wheat middlings 


115 
116 
117 


20 
15 
12 


10 
12 
10 


2 
16 
13 


10 








42 
43 
35 


10.5 
14.3 
11.7 


12.0 


42,000,000 










420,0(0 










420,000 













41 




118 
119 
120 


12 
8 
9 


14 
2 

4 


8 

6 

10 











34 
16 
23 


8.5 
5.3 

7.7 


7.3 


12,000,000 












120,000 












120,000 














4 17 


Partly decomposed 
cow feces 


121 
122 
123 


5 
6 
6 


4 
12 



5 

7 
18 








14 
64 
38 


4.7 

10.7 

7.6 


8.3 


324,000,000 




3 
12 


16 
2 


20 




3,240,000 
3,240,000 










4ft 


Fiesh cow feces 


124 
125 
126 


10 
14 


12 



7 
5 


15 
12 






44 
31 


11.0 

7.7 


9.4 


81,000,000 










810,000 










810,000 
























-1 t 


Muck . . . 


127 
128 
129 


4 

8 
1 



12 

8 


o 
o 

7 




o 

2 








4 
20 
18 


1.0 
5.0 

4.5 


3.5 


24,000,000 












240,000 










240,000 












45 




130 
131 
132 


21 
17 
12 


10 
14 
22 


7 
16 

8 


6 
2 








44 
49 
42 


11.0 
12.2 
14.0 


12.3 


2,000,000 












20,000 










20,000 













66 Martin J. Frucha 

In this test a known number of organisms was introduced into each 
flowerpot at the time of inoculation, in order to trace the relation between 
the number of nodules developed and the number of organisms used for 
inoculation. It was hoped that in this way some means might be found 
of measuring not only the loss, but also the degree, of infecting power. 
From tlic data in table 15 it is seen thai absolutely no relation exists 
between the number of organisms introduced into the flowerpots and the 
number of nodules that developed, so that the only measure of infecting 
power is the presence or the absence of nodules. This, however, does not 
measure the degree of infecting power, but only its presence or absence. 

The average number of nodules per plant produced by each culture, as 
shown in table 15, are rearranged in numerical order in table 16. It is 
found on comparing tables 13 and 16 that the results of the third test of 
infecting power show a general agreement with the results of the second test. 

If it is considered that the presence of three or more nodules per plant 
indicates that the culture was efficient, the following cultures are seen to 
have lost their efficiency : 

Soy bean hay 
Soy bean roots 
• Canada field pea hay 
Canada field pea roots 
Medium 334 + 10 per cent cane sugar 
Medium 334 + 40 per cent cane sugar 
Medium 334 + 0.2 per cent phloroglucin 
Medium 334 -f- 1 per cent salicin 
Medium 334 + 0.5 per cent amygdalin 
Medium 334 -f- 0.5 per cent resorcin 
Medium 334 -f 0.5 per cent potassium oxalate 
Medium 334 + 5 per cent Witte's peptone 

The cultures that had any living organisms at the time of inoculation 
produced nodules practically in all cases in the three tests. The cultures in 
which no living organisms were found produced no nodules, or only a few 
unevenly distributed. It must be remembered that a certain amount of 
contamination may occur, and that the method of determining the presence 
of living organisms by plate cultures is not absolutely accurate. The cul- 
tures in this test were ten weeks older than those in the second test. The 
results show that the nodule-bacteria cultures can be kept for at least 
twenty weeks, and the bacteria will still be efficient in producing nodules. 



Studies of Bacillus Radicicola of Canada Field Pea 67 

TABLE 16. Average Number or Nodules per Plant „ Third Test, Arranged in 

Numerical Order 



Medium 



Sandy soil 

Wheat middlings 

Medium 334 + 2 per cent glycerin .... . 

Medium 334 + 0.1 per cent asparagin 

Wheat bran 

Medium 334 + 1 per cent Merck's peptone 
Medium 334 + 0.1 per cent potassium nitrate. 
Medium 334 + 1 per cent Witte's peptone. 

Fresh cow feces 

Medium 334 -f- 0.6 per cent calcium nitrate. 

Cornstarch 

Medium 334 + 0.5 per cent potassium citrate. 
Medium 334 + 0.2 per cent calcium nitrate. . . 

Partly decomposed cow feces 

Medium 334 + 2 per cent Witte's peptone. '.'.' . 

Medium 334 + 2 per cent mannite 

Corn meal 

Compost 

Medium 
Medium 
Medium 
Sawdust 
Medium 
Medium 
Medium 
Medium 
Medium 
Medium 
Medium 
Muck... 



334 + 0.1 per cent calcium nitrate. . . 
334 + 0.6 per cent potassium nitrate. 
334 + 5 per cent gelatin 



334 + 2 per cent galactose 

334 + 0.2 per cent Merck's peptone . 

334 + 2 per cent lactose 

334 + 2 per cent cane sugar 

334 + 2 per cent cane sugar 

334 + 2 per cent dextrose 

334 + 0.2 per cent potassium nitrate 



Medium.334 + 10 per cent cane" sugar 

Medium 334 + 20 per cent cane sugar 

Medium 334 + 0.2 per cent phloroglucin . . . 

Medium 334 + 10 per cent cane sugar 

Medium 334 + 0.5 per cent potassium oxalate . 

Medium 334 + 5 per cent Witte's peptone 

Medium 334 + 40 per cent cane sugar 

Medium 334 + 0.5 per cent resorcin 

Medium 334 + 1 per cent salicin 

Medium 334 + 0.5 per cent amygdalin 

Canada field pea hay 

Canada field pea roots 

Soy bean hay 

Soy bean roots 



Organisms 
living or 
not livin ; 



+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 



Number of 
nodules 
per plant 



12.3 

12.0 

10.5 

10.0 

9.8 

9.5 



8.6 
8.3 
7.9 



6.6 



+ 


5.9 


+ 


5.9 


+ 


5.3 


+ 


4.8 


+ 


4.5 


+ 


4.4 


+ 


3.9 


+ 


3.5 


+ 


3.1 


+ 


3.0 





1.6 


+ 


1.4 





0.3 





0.1 



















































* + indicates living organisms; indicates no living organisms. 



68 Martin J. Prucha 

EXPERIMENT 13 
INFLUENCE OF MEDIA 300, 310, 335, AND 400 

The organism used for this experiment was propagated on medium 335, 
and had been kept in the laboratory for two years and three months, 
where it was exposed to diffused light and transfers had been made at 
intervals. When the experiment was started the organism readily produced 
nodules on the plants, showing that its infecting power had not been lost. 

The plan of the experiment was to propagate the organism on both 
the nitrogen-free and the nitrogenous media for a period of time, and then 
to test these cultures for nodule production. The following procedure 
was adopted: Media 300, 310, 335, and 400 were introduced into test 
tubes, sterilized, and sloped. Four slopes from each of these media were 
inoculated with the same culture of the organism. A few days later a 
second set of four slopes from each medium was inoculated, and so on. 
Nine such sets of agar slopes from each of the four media were inoculated 
in one hundred and fifteen days. The four agar slopes of the same medium 
in each set were inoculated from one of the four slopes of the same medium 
from the previous set, so that on all the slopes of the same medium in 
the nine sets the organism was under the influence of the same media for 
one hundred and fifteen days. The age of the agar slopes, however, 
differed in the respective sets. When the ninth set was inoculated, four 
additional tubes of media 300, 310, and 400 were inoculated with a culture 
of the organism which up to that time had been propagated on nitrogen- 
free medium 335. These test-tube cultures are designated in table 17 
as set X. They were fifteen days old and had been under the influence 
of nitrogenous media for only fifteen days when tested for infecting power. 
In order to prevent the effects of drying, melted paraffin was poured on 
the cotton plugs after a good growth had developed. All the cultures 
were kept at room temperature. 

The infecting power of these agar-slope cultures was tested by inoculating 
Canada field peas. The method of growing and examining the plants was 
the same as in experiment 12. In inoculating the plants, the growth from 
each agar slope was introduced into 100 cubic centimeters of sterile water, 
the number of organisms in this was determined by the plate method, and a 
definite quantity of this infusion was poured over the seeds in each flowerpot. 

Unfortunately, an accident happened to a large number of the flowerpots 
in the greenhouse, and consequently the data are not complete. As far 
as they could be obtained, they are summarized in table 17: 



Studies of Bacillus Radicicola of Canada Field Pea 



69 







TABLE 


17. Influence of Media 


300, 


310, 


335, and 400 




9 

5 

"3 

o 

"o 

u 
o 

P 


<o 
"o 

M 

09 

P 
3 
2 


P 
a 
•3 


a 

Oj 
J3 

is 

0J 3 

M O 

3 "43 

§1 

■sj 

"In u 

03^ 
0) 

.SB 

o 

to 

< 


OJ 

is 

o 

qa 

"o 

u 

oj 
j2 m 

go 

>. 

o 

a 
u 

o 

JQ 

o3 

h3 


2 =4 

"o a 

l'3'S, 
S3 


a 

tn 

oj 

"3 

1 


O 

H 


OJ 

3° 

T3 Q, 

2 " 

3 - 

is 

V. O 

oca 

A 

2 § 

P C 
3'- 

°3- 

••f 

54 u 
CO 




OJ — 
oj 3 

0^ 

•as 

3 

fl a 

Sffl 
fc! 53 
4>"o. 

< 


m 

1 

'3 

c3 
bd 

O '_ 

.53 

> 5J 


3 
13 

>> 



03 

(- 

o 

■i 

►J 


03 


03 

a 

c4 


co 

a 

a 

s 


a 

03 

s 


3 

s 


CO 

3 

03 

S 

6 


J 0) 

J3 

°.a 

il 


1 


1 


335 


115 


1 
2 
3 



13 
25 


6 
15 
16 


14 

12 

2 


7 

7 


6 
8 


39 
55 
43 


6.5 
11.0 
14.3 


9.8 


300,000,000 














? 


1 


300 


115 


4 
5 

6 

































1 1 



J 























3 


I 


400 


115 

1 


7 

8 
9 










































I 1 



|{ 


1 


4 


10 

II 335 110 < 11 

12 


12 
6 


13 

8 


8 
8 


8 
5 


5 

5 


"2 


46 
34 


9.2 
5.7 


1 

7.3 


350,000,000 
























5 


II| 300 110 


13 

14 
15 






















n 







































' 6 


II 400 110 


16 
17 
18 
























6 


' ' 4 



4 
6 




0.7 

1.2 


0.6 





7 


III 


335 ! 105 


19 

20 

21 


8 
9 

8 


9 
20 
10 


12 
6 

7 


20 14 

7 4 
8| 




63 
46 
33 


12.6 
9.2 
6.6 


9.5 


360,000,000 


8 


III 


300 105 | 


22 
23 
24 




































1 1 



J 





9 


III 


400, 105 | 


25 

28 
27 






















o 
















il 

1 
j J 





10 


III 


310 105] 


2S 
29 
30 


12 
14 

7 


5 
16 
10 


15 
6 
6 


4 
18 
1? 


10 

8 


15 
13 


61 
75 
35 


10.2 I 

12.5 10.7 

8.7 J 


144,000,000 










11 


IV 


400 98 


31 
32 
33 





2 


oj 






2 


0.5 1 










0.5 


90,000 






















........ 












12 


VI 


f 
335 81 

{ 


34 
35 

36 


13 

4 

14 


1 

2 
10 


4 

14 

6 


17 
8 



6 
2 
8 


"5 
3 


41 
35 

41 


8.2; 1 

5.8, 6.9 

6.8! J 


180,000,000 


13 


VI 


300 81 

! 1 


37 
38 
39 





































































14 


VI 


400 81 


40 

41 
42 


10 

12 


11 

20 
10 


12 
6 
6 


7 
7 



13 


"8 


40 
54 
34 


8.0| ] 

9.0 8.5 

8.5 J 


1,000,000 










15 


VI 


f 
310, 81 | 


43 
44 
dR 


20 


15 


8! 






43 


10.7 \ 










10.7 


540,000,000 





































70 



Martin J. Prucha 















TABLE 


17 (concluded) 












0) 

t- 

S 
3 

= 






a 

o 

A 

» 9 

M 

3 '5 

— 8! 

g 
*-. o 

°.s 

>«2 


a 


"o 

M 

oj 

I* 

3 P. 


12 ° 

2 * 

3 a> 

"3 a 

* ° - 

g o> cj 


a 

"3 

§ ! 

(S 



03 
AM 


01 

1° 

3 <L> 

„_, 
ow 

.0 0> 

3d 


u 0> 

t, 
0.3 

11 

Ov 1> 

si 


1 

22 
'3 

03 
M 
u 

O Q) 

m3 
•9-3 


a 
















fc 


'o 




°3^ 
















3 ° 




3 

a 


g 


o 

■a 


oj 
£ 

3 


£ 

3 

■5 


.93 

IB 
M 
"3 


03 

O 

03 
h5 


5 


IN 

a 

03 

5 


CO 

a 

a 


a 


a 

03 




a 

03 

s 


*o3 
O 

H 


m a 
> a 


01 


fc-o 
11 










46 


12 





6 


8 





16 


42 


7.0 


1 




10 


VII 


335 


61 


47 


16 


7 


15 


6 


20 




64 


12.8 


9.8 


396,000,000 










48 


3 


20 





6 


22 


9 


60 


10.0 


1 






VII 


400 


61 | 


49 
50 
51 


2 
5 


10 
5 


4 
12 








16 
38 


5.3 
9.5 


7.7 




17 


16 






10,000,000 






































52 
































18 


VII 


310 


01 


53 










































54 








































55 





10 


8 


9 







27 


5.4 


1 




10 


VIII 


335 


49| 


56 

57 


8 
8 


5 
10 



9 








13 
34 


4.3 

8.5 


6.2 


216,000,000 




7 


















VIII 


300 


49] 


58 
59 


7 
6 


9 

7 










16 
66 


8.0 
11.0 


11.2 




20 


18 


7 


16 


12 


16,000,000 










00 


6 


13 


30 


4 






53 


13.2 


1 




















61 


8 





2 


18 







28 


5.6 


1 




21 


VIII 


400 


49 | 


62 


12 


13 


4 


4 


14 




47 


9.4 


6.8 


252,000,000 










63 


4 





1 


6 


16 




27 


5.4 


J 






VIII 


310 


49 


64 
65 
00 




6 

12 


6 
10 

2 


25 
6 









31 
27 
36 


10.3 
6.7 
7.2 


1 




?,?! 


5 
10 








7.8 


8,000,000 




12 














07 


10 


6 


5 


2 


8 




31 


6.2 


I 




23 


IX 


335 


15 


68 


4 


2 








4 


10 


2.0 


4.5 


576,000,000 










69 


8 


14 












22 


5.5 


J 














IX 


300 


15 | 


70 
71 


12 
18 


35 



23 




13 






70 
44 


15.0 
7.3 


8.5 




24 


5 


8 


4,320,000,000 










72 





7 





2 


5 




14 


2.8 


J 






IX 


4C0 


15 


73 
74 



10 


5 

4 


8 
4 



2 






13 
37 


3.2 
6.2 


5.8 




25 


7 


10 


2,592,000,000 










75 


7 


18 











25 


8.3 




















76 


9 


5 


4 


7 








25 


4.2 


1 




w; 


IX 


310 


15 


77 
78 


8 
12 



8 


4 
8 


7 
15 






19 
55 


4.7 
9.2 


\ 6.2 


3,240,000,000 







12 












79 


5 








12 


6 


23 


4.6 


I 




27 


X 


300 


15 


80 


4 


7 


30 


20 


12 




73 


14.6 


7.1 


180,000,000 










81 








3 


3 


5 


.... 


11 


2.2 


J 












82 


2 


15 


2 


8 


6 




33 


6.6 


! 1.. 




28 


X 


400 


15 


83 


5 


9 


1 


6 


6 


8 


35 


5.8 


972,000,000 










84 


6 


8 







6 


8 


53 


8.8 


J 












85 


6 








7 


7 


4 


24 


4.0 


1 




29 


X 


310 


15 


86 

87 



12 



25 









7 


10 


17 
37 


2.8 
12.3 


5.2 


1,152,000,000 

















Studies of Bacillus Radicicola of Canada Field Pea 



71 



Results 

The nature and the composition of the four media used in experiment 
13 are given in table 1, page 11. No nitrogen in any form was added to 
medium 335. The other three media received 0.3 per cent of Liebig's 
beef extract, and, in addition to this, media 300 and 310 received 1 per 
cent and medium 400 received 2 per cent of Witte's peptone. Medium 
335 contained 2 per cent of cane sugar, and medium 310 contained 2 per 
cent of dextrose. 

As seen from table 17, the age of the individual cultures varied between 
fifteen days and one hundred and fifteen days; but the organisms in all 
the cultures, except 27, 28, and 29, had been under the influence of their 
respective media for one hundred and fifteen days. Cultures 27, 28, and 
29 were fifteen days old and had been under the influence of their respec- 
tive media for only fifteen days, having been grown previously on medium 
335. 

The influence of these four media on the organism is shown in table 17. 
In order to bring out the relations more clearly, a part of these data are 
rearranged in table 18: 



TABLE 18. Influence of Age of Cultures on the Given Media 



Laboratory number of culture 



Culture 
medium 



Age of 
culture 
(days) 



Average 

number 

of nodules 

per plant 



Number of 

living 
organisms 
in culture 



23 
19 

hi 

12 

7 

4 

1 

24 
20 
13 

8 
5 
2 

25 
21 

17 
14 



335 
335 
335 
335 
335 
335 
335 

300 
300 
300 
300 
300 
300 

400 
400 
400 
400 



15 

49 

61 

81 

105 

110 

115 

15 

49 

81 

105 

110 

115 

15 
49 
61 

81 



5.8 
6.8 

7.7 
8.5 



576,000,000 
216,000,000 
396,000,000 
180,000,000 
360,000,000 
350,000,000 
300,000,000 

4,320,000,000 
16,000,000 





2,592,000,000 

252,000,000 

10,000,000 

1,000,000 



72 



Martin J. Prucha 



TABLE 18 (concluded) 



Laboratory number of culture 


Culture 
medium 


Age of 
culture 
(days) 


Average 

number 

of nodules 

per plant 


Number of 

living 
organisms 
in culture 


11 


400 
400 
400 
400 

310 
310 
310 
310 
310 

300 
400 
310 


98 
105 
110 
115 

15 
49 
61 
81 
105 

15 
15 
15 


0.5 


0.6 


6.2 
7.8 


10.7 
10.7 

7.1 
7.1 
5.2 


90,000 


9 





6 





3 





26 


3,240,000,000 


22 


8,000,000 


18 





15 


540,000,000 


10 


144,000,000 


27 


180,000,000 


28 


972,000,030 


29 


1,152,000,000 







The organism had been propagated and kept on medium 335, a non- 
nitrogenous medium, for over two years before this experiment was started. 
Several tests of infecting power were made during that time. This me- 
dium was used also in experiment 12. As far as could be judged by all 
these tests, the infecting power of the organism remained constant when 
propagated and kept on this medium. Therefore the results shown in 
experiment 13 as to the infecting power of the cultures on medium 335 
are taken to represent the normal infecting power of the organism, and 
serve as a basis for comparison of the infecting power of the cultures on 
the other media. 

Of the seven cultures on medium 300, a nitrogenous medium, three 
cultures — namely, 20, 24, and 27 — produced nodules. Cultures 24 and 
27 were fifteen days old and culture 20 was forty-nine days old. The 
organisms in cultures 20 and 24 were under the influence of medium 300 
for one hundred and fifteen days, and culture 27 for only fifteen days. 
These three cultures appeared as effective as those on medium 335. The 
remaining cultures on medium 300 were from eighty-one to one hundred 
and fifteen days old and did not produce any nodules, apparently having 
lost their power of infection. 

Similar results were obtained with the nitrogenous medium 400. Of 



Studies of Bacillus Radicicola of Canada Field Pea 73 

the nine cultures on this medium, five produced nodules, two were doubtful, 
and two produced no nodules. Culture 28 was fifteen days old and had 
been under the influence of this medium for only fifteen days; while culture 
25 was fifteen days old and had been under the influence of this medium 
for one hundred and fifteen days. No appreciable difference in the in- 
fecting power of these two cultures was observed. 

Medium 310 is the same as medium 300 except that it contains 2 per 
cent of dextrose. Five of the six cultures on this medium produced 
nodules; one did not, and that was not the oldest culture. Comparing 
these results with those from medium 300, it seems as if the dextrose 
protected the infecting power of the organism on this medium. 

In the last column of Table 18 the number of living organisms in each 
culture is given, and in the next to the last column the number of nodules 
per plant produced by each culture is given. It is seen that in all cases 
in which no living organisms were found in a culture no nodules were 
produced by that culture, and that the cultures in which living organisms 
were present produced nodules. Culture 6 is the only exception, but in 
this case only two plants out of sixteen had four and six nodules, respec- 
tively, and the size and location of these nodules pointed to later con-, 
tamination. 

According to the above data, Bacillus radicicola of Canada field pea 
does not lose its infecting power when propagated on medium 300 and 
400 for one hundred and fifteen days. Some of the cultures on these two 
media did lose their infecting power, but in all cases these were the older 
cultures. This loss in efficiency is due to death of the culture, and death is 
induced by nitrogenous media after a considerable duration. 

SUMMARY 

1. The causal organism in the case of Canada field pea nodules is Bacillus 
radicicola. Its flagella are peritrichic, and eight was the largest number 
found. Its group number is B. 222.2322033. 

2. Nodules developed both in light and in darkness. A larger number 
of nodules, however, developed in darkness. 

3. Nodules developed readily both in the soil extract and in synthetic 
nutrient solutions in which the nitrates were either omitted or replaced 
by chlorides. The nodules continued to increase in number as long as 
the plants continued to grow. 



74 Martin J. Prucha 

4. In a full nutrient solution containing nitrates a few nodules may de- 
velop immediately after inoculation, but a subsequent continual develop- 
ment of nodules seems to be inhibited. 

5. No nodule development took place in nutrient solutions in which 
the individual essential elements were omitted, except in the case of ni- 
trogen. 

6. In sandy soil a moisture content of 20 to 40 per cent was more favor- 
able for nodule development than lower or higher percentages. 

7. The addition of KN0 3 , Ca(N0 3 ) 2 , NH 4 C1, FeCl 3 KC1, or peptone to 
sandy soil in the proportion of \ gram of the salts to 300 grams of the soil, 
air-dry, had an inhibiting effect on nodule development on Canada field 
peas. The addition of MgS0 4 , KH 2 P0 4 , Ca(H 2 P0 4 ) 2 , and tannic acid, 
especially at the lower concentrations, in 300 grams of the soil, had a 
beneficial effect on nodule development on Canada field peas. 

8. Nutrition markedly influences the morphology of the nodule or- 
ganisms. 

9. The addition of 1 cubic centimeter or more of a normal solution 
of HC1 to 10 cubic centimeters of agar medium 334, 335, or 337 was in- 
jurious to the vitality, and therefore to the infecting power, of the alfalfa- 
nodule organism. The addition of 2 cubic centimeters or less of a normal 
solution of NaOH to 10 cubic centimeters of each of the above media 
seemed to be slightly beneficial to the vitality and the infecting power 
of this organism. 

10. The organism of Canada field peas produced no visible growth in 
medium 334 when the following substances were added: levulose 2 per 
cent, phloroglucin 0.2 per cent, potassium oxalate 0.5 per cent, potassium 
citrate 0.5 per cent, Witte's peptone 5 per cent, Merck's peptone 
3 per cent. 

11. The organism multiplies readily in some soils and in various sub- 
stances; as many as 10,000,000,000 organisms per gram developed in wheat 
bran and in ground peas. 

12. The infecting power of B. radicicola of Canada field pea was not 
affected after the organism had been kept on medium 335 for two years 
and a half in the laboratory, the culture being transferred once each month. 

13. The infecting power of the organism was not appreciably influenced 
by the various media. All the cultures in which living nodule-forming 
organisms were found at the time of trial produced nodules. 



Studies of Bacillus Radicicola of Canada Field Pea 75 

14. In some media and under certain conditions the organisms died 
much sooner than in other media. The nitrogenous media did not seem 
to influence the infecting power of the organism. 

15. It is not difficult to determine whether or not a given culture can 
produce nodules, but there is no accurate method of measuring the slight 
variations in infecting power that may exist in the different cultures. 

GENERAL DISCUSSION 

The definition of virulence as given by various authors in the medical 
texts is not clear, but in general the word is defined as meaning the power 
of microorganisms to invade and multiply in the tissues of a host and 
cause some injury or disease to the host. It was found by Peirce (1902), 
Fred (1911), and others, that the cells in the nodule are injured by the 
nodule-forming organism and become abnormal, and that for this reason 
the relation between the microorganism and the legume is a parasitic 
one. Furthermore, a nodule on a leguminous plant is a swelling, a hy- 
pertrophic formation; and morphologically speaking, it is an abnormality, 
or a form of disease. Nodule formation, therefore, may be considered as 
of a pathological nature. But, it may be asked, which is the normal root 
of the legume — the one with the nodules, or the one without the nodules, 
or both? Whatever the morphological and cytological evidence may be, 
one well-established fact stands out; namely, that leguminous plants are 
benefited by the presence of nodules. No positive evidence has been 
produced thus far to show that the microorganism which causes nodules 
is injurious to leguminous plants. The nodule-forming organism pen- 
etrates the root tissue, multiplies therein, and apparently derives its 
necessary food therefrom; and in return for this it enables the plant to 
obtain a certain amount of nitrogen. There is, therefore, a mutual and 
beneficial exchange and the relation is symbiotic. 

The primary object of the experiments reported in this investigation 
was to determine whether -the power of the nodule-forming organism to 
cause nodules is easily altered by artificial media. The nodule-forming 
organism of Canada field pea was chosen for this purpose, and the results 
apply only to that organism. It is probable that the organisms from 
other legumes might have given different results. Since the value of the 
investigation depended so much on the securing of a pure culture, the 
various precautions, as previously indicated, were adopted for this purpose. 



7G Martin J. Prucha 

The fact that a pure culture is obtained from a nodule and resembles in 
its cultural characteristics the true nodule-forming organism is not a suffi- 
cient proof that such an organism is the nodule-forming organism. De' 
Rossi (1907), in his investigation, emphasizes this point, and unques- 
tionably the information on the general subject of nitrogen fixation by 
leguminous plants has been colored by results from experiments in which 
some organism other than the nodule-forming organism was employed. 

As stated on page 59, the number, size, and location of the nodules 
on the roots are probably influenced by a number of factors. Since the 
number of nodules produced on the plants in a given time was used 
as the measure of the infecting power of the culture with which the 
plants were inoculated, it seemed advisable to study the influence of 
several factors and thus to determine whether Canada field peas readily 
form nodules under the conditions that it was planned to use in Part III. 
The results of these experiments in Part II tend to point to the conclusion 
that, in general, the conditions favoring the normal development of plants 
favor also the development of nodules. An exception to this is found in 
the fact that the presence of nitrates tends to inhibit the development 
of nodules, and at the same time favor the normal development of the 
plants. No satisfactory explanation for this phenomenon has as yet been 
given. The plants appear not to be injured by the presence of nitrates, 
and neither do the nodule-forming organisms seem to be injured when 
propagated on a medium in which nitrate is present (see experiment 13). 
The explanation that the plants are made more vigorous when supplied 
with nitrogen, and can more readily resist the invasion of the microorgan- 
isms, cannot be taken seriously. It might be noted here that the root 
system of Canada field peas grown in Pfeffer's nutrient solution appears 
normal, tending to become slightly brownish; but when the nitrate in the 
same solution is replaced by the chloride of the same metal, the root 
system becomes larger, the roots being more numerous and longer. Con- 
sequently, the rate of growth of the root tissue is accelerated by the ab- 
sence of nitrates. Whether this somewhat rapid growth of the root 
tissue has any relation to nodule formation is not known. It is highly 
probable that a biological factor also influences the development of nodules. 
The microbial flora of the soil or of the solution in which the plants are 
grown is undoubtedly influenced by the composition of that soil or solution. 
The microorganisms that thrive best in a highly nitrogenous soil or solu- 



Studies of Bacillus Radicicola of Canada Field Pea 11 

tion, and the products of their metabolic activities, may exercise an in- 
jurious influence on the nodule organisms and thus prevent them from 
multiplication and distribution through the soil. 

The influence of artificial media on the power of Bacillus radicicola to 
cause nodules is of considerable importance, in view of the fact that 
inoculation for leguminous crops with pure cultures is extensively prac- 
ticed and the pure cultures have to be propagated on some media. Frank 
(1S99), in commenting on the low efficiency of nitragin, suggested that 
probably the medium (gelatin) on which the cultures were propagated 
and kept was not favorable for the organisms, and that this might be the 
cause of the low efficiency of nitragin. Hiltner (1900) finally substituted 
liquid media for the gelatin, and was able to obtain better results from 
the legume inoculation. Suchting (1904) found that not only the gelatin 
media are injurious to the bacteria — a point shown by Hiltner — -but 
the agar media also may be unfavorable. Moore (1905) made the ob- 
servation that the nodule-forming bacteria increase most rapidly on a 
medium rich in nitrogen, but that the resulting growth is usually very 
much reduced in infecting power. Lewis and Nicholson (1905), on the 
other hand, state that the presence or absence of nitrogen in the culture 
media is not the determining factor in maintaining the activity of the 
germ. 

A reasonable conclusion from the investigations mentioned above 
seems to be that some artificial media are more favorable than others 
for the propagation of B. radicicola, and that the amount of growth is 
not always directly proportional to the nodule-producing efficiency of 
the organism. With this conclusion the experiments in Part III are in 
accord. In addition to this, the experiments point to the conclusion 
that B. radicicola of Canada field pea does not possess " virulence " in 
a pathological meaning of the word. The ability to cause nodules is so 
closely bound up with the general vitality of the bacteria that our means 
and methods cannot detect any variations, if such there are, in their 
nodule-producing ability. The writer's opinion is that every living 
nodule-producing organism in a vigorous condition, will, if given a chance, 
cause nodule development no matter on what kind of media it has been 
propagated. The propagation of the organisms on different media does 
not measurably affect their nodule-producing efficiency. The organisms 
die sooner on some media than on others, and the loss of the nodule- 



78 Martin J. Prucha 

producing efficiency of the cultures is due to the dying-out of the organ- 
isms. Whether the injurious agent is some ingredient of the media, or 
lack of a proper nutrient, or the accumulation of the metabolic products, 
has not been determined. The observation that a cop ous growth is 
usually of reduced nodule-producing efficiency suggests the last as the 
probable, or at least a partial, explanation. 

When one examines leguminous plants grown under favorable conditions, 
one finds that each plant has a limited number of nodules and that the 
number and the size of the nodules vary on the different plants. Hiltner 
(1900), holding the relation between B. radicicola and the leguminous plants 
to be of a pathological nature, concluded from his experiments that the 
variableness of the infecting power of the nodule-forming bacteria is the 
limiting factor which determines the number and size of the nodules under 
otherwise favorable conditions. Such ting (1904), in trying to account for 
the limited number of nodules on each plant, emphasized the resistance 
of the plants against the invasion of the bacteria. His explanation is 
as follows: " In contrast to Hiltner's immunity theory, I am of the opinion 
that the nodule formation and their number are regulated by the relation 
between the antibodies in the plant and the infecting substance of the 
bacteria." 7 

These explanations do not appear to the writer to explain the conditions. 
No pathological explanation can account for a limited number of nodules. 
The physical condition and the chemical composition of the soil, the 
amount of moisture, and the microbial flora of the soil, are some of the 
important factors that interfere, directly or indirectly, with the coming 
together of B. radicicola and the roots of the plants. The stage in the 
development of the plant, the rate of growth of the roots, the number 
of root hairs, the character of the root tissue, and other factors, may also 
play a part in limiting the development of nodules. 



ACKNOWLEDGMENTS 

It is with pleasure that the writer here acknowledges his indebtedness 
to Professors Lewis Knudson and B. M. Duggar for much helpful advice 
and criticism throughout the progress of this work. 

7 Translation from the original German. 






Studies of Bacillus Radicicola of Canada Field Pea 79 



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19 1 2 Beitrage zur morphologie und biologie der knollchenbakterien 
der leguminosen. Centbl. bakt. 2:32:97-137. 

Submitted for publication, June, 1914. 



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